Modulation of hepatitis B virus (HBV) expression

ABSTRACT

Disclosed herein are antisense compounds and methods for decreasing HBV mRNA, DNA and protein expression. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate HBV-related diseases, disorders or conditions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 13/452,703, filed Apr. 20, 2012, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/478,040, filed Apr. 21, 2011; U.S. Provisional Patent Application No. 61/478,038, filed Apr. 21, 2011; U.S. Provisional Patent Application No. 61/596,690, filed Feb. 8, 2012; and U.S. Provisional Patent Application No. 61/596,692, filed Feb. 8, 2012, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0175USC1.5T25.txt created Dec. 18, 2013, which is approximately 276 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

In certain embodiments provided are methods, compounds, and compositions for inhibiting expression of hepatitis B virus (HBV) mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate HBV-related diseases and disorders.

BACKGROUND

Hepatitis B is a viral disease transmitted parenterally by contaminated material such as blood and blood products, contaminated needles, sexually and vertically from infected or carrier mothers to their offspring. It is estimated by the World Health Organization that more than 2 billion people have been infected worldwide, with about 4 million acute cases per year, 1 million deaths per year, and 350-400 million chronic carriers (World Health Organization: Geographic Prevalence of Hepatitis B Prevalence, 2004. http://www.who.int/vaccines-surveillance/graphics/htmls/hepbprev.htm).

The virus, HBV, is a double-stranded hepatotropic virus which infects only humans and non-human primates. Viral replication takes place predominantly in the liver and, to a lesser extent, in the kidneys, pancreas, bone marrow and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64: 2000; 51-68.). Viral and immune markers are detectable in blood and characteristic antigen-antibody patterns evolve over time. The first detectable viral marker is HBsAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA.

Titers may be high during the incubation period, but HBV DNA and HBeAg levels begin to fall at the onset of illness and may be undetectable at the time of peak clinical illness (Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med. 350: 2004; 1118-1129). HBeAg is a viral marker detectable in blood and correlates with active viral replication, and therefore high viral load and infectivity (Hepatitis B e antigen—the dangerous end game of hepatitis B. N Engl J Med. 347: 2002; 208-210). The presence of anti-HBsAb and anti-HBcAb (IgG) indicates recovery and immunity in a previously infected individual.

Currently the recommended therapies for chronic HBV infection by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) include interferon alpha (INFα), pegylated interferon alpha-2a (Peg-IFN2a), entecavir, and tenofovir. The nucleoside and nucleotide therapies, entecavir and tenofovir, are successful at reducing viral load, but the rates of HBeAg seroconversion and HBsAg loss are even lower than those obtained using IFNα therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT), and adefovir are also used, but for nucleoside/nucleotide therapies in general, the emergence of resistance limits therapeutic efficacy.

Thus, there is a need in the art to discover and develop new anti-viral therapies. Additionally, there is a need for new anti-HBV therapies capable of increasing HBeAg and HBsAg seroconversion rates. Recent clinical research has found a correlation between seroconversion and reductions in HBeAg (Fried et al (2008) Hepatology 47:428) and reductions in HBsAg (Moucari et al (2009) Hepatology 49:1151). Reductions in antigen levels may have allowed immunological control of HBV infection because high levels of antigens are thought to induce immunological tolerance. Current nucleoside therapies for HBV are capable of dramatic reductions in serum levels of HBV but have little impact on HBeAg and HBsAg levels.

Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of HBV expression (See U.S. Patent Publication Nos. 2008/0039418 and 2007/0299027). Antisense therapy differs from nucleoside therapy in that it can directly target the transcripts for the HBV antigens and thereby reduce serum HBeAg and HBsAg levels. Because of the multiple, overlapping transcripts produced upon HBV infection, there is also an opportunity for a single antisense oligomer to reduce HBV DNA in addition to both HBeAg and HBsAg. Therefore, antisense technology is emerging as an effective means for reducing the expression of certain gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of HBV.

SUMMARY

Provided herein are methods, compounds, and compositions for modulating expression of HBV mRNA and protein. In certain embodiments, compounds useful for modulating expression of HBV mRNA and protein are antisense compounds. In certain embodiments, the antisense compounds are antisense oligonucleotides.

In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, HBV mRNA levels are reduced. In certain embodiments, HBV DNA levels are reduced. In certain embodiments, HBV protein levels are reduced. In certain embodiments, HBV antigen levels are reduced. In certain embodiments, HBV s-antigen (HBsAg) levels are reduced. In certain embodiments, HBV e-antigen (HBeAg) levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.

Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, such HBV related diseases, disorders, and conditions are liver diseases. In certain embodiments, such liver diseases, disorders, and conditions includes jaundice, liver cancer, liver inflammation, liver fibrosis, inflammation, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and liver disease-related transplantation. In certain embodiments, such HBV related diseases, disorders, and conditions are hyperproliferative diseases, disorders, and conditions. In certain embodiments such hyperproliferative diseases, disorders, and conditions include cancer as well as associated malignancies and metastases. In certain embodiments, such cancers include liver cancer and hepatocellular cancer (HCC).

Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common. Certain risk factors and causes for development of liver disease or a hyperproliferative disease include growing older; tobacco use; exposure to sunlight and ionizing radiation; contact with certain chemicals; infection with certain viruses and bacteria; certain hormone therapies; family history of cancer; alcohol use; and certain lifestyle choices including poor diet, lack of physical activity, and/or being overweight. Certain symptoms and outcomes associated with development of a liver disease or a hyperproliferative disease include but are not limited to: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine.

In certain embodiments, methods of treatment include administering a HBV antisense compound to an individual in need thereof. In certain embodiments, methods of treatment include administering a HBV antisense oligonucleotide to an individual in need thereof.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

DEFINITIONS

Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxy-ethyl modification at the 2′ position of a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanosyl ring other than H or OH. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications.

“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.

“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within ±7% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of HBV”, it is implied that the HBV levels are inhibited within a range of 63% and 77%.

“Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits.

“Active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to HBV is an active pharmaceutical agent.

“Active target region” means a target region to which one or more active antisense compounds is targeted. “Active antisense compounds” means antisense compounds that reduce target nucleic acid levels or protein levels.

“Acute hepatitis B infection” results when a person exposed to the hepatitis B virus begins to develop the signs and symptoms of viral hepatitis. This period of time, called the incubation period, is an average of 90 days, but could be as short as 45 days or as long as 6 months. For most people this infection will cause mild to moderate discomfort but will go away by itself because of the body's immune response succeeds in fighting the virus. However, some people, particularly those with compromised immune systems, such as persons suffering from AIDS, undergoing chemotherapy, taking immunosuppressant drugs, or taking steroids, have very serious problems as a result of the acute HBV infection, and go on to more severe conditions such as fulminant liver failure.

“Administered concomitantly” refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.

“Agent” means an active substance that can provide a therapeutic benefit when administered to an animal. “First Agent” means a therapeutic compound described herein. For example, a first agent can be an antisense oligonucleotide targeting HBV. “Second agent” means a second therapeutic compound described herein (e.g. a second antisense oligonucleotide targeting HBV) and/or a non-HBV therapeutic compound.

“Amelioration” refers to a lessening of at least one indicator of the severity of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, F_(ab) region, and F, region.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, snoRNAs, miRNAs, and satellite repeats.

“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar” means a furanose ring modified by the bridging of two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.

“Body weight” refers to an animal's whole body weight, inclusive of all tissues including adipose tissue.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

“Chronic hepatitis B infection” occurs when a person initially suffers from an acute infection but is then unable to fight off the infection. Whether the disease becomes chronic or completely resolves depends mostly on the age of the infected person. About 90% of infants infected at birth will progress to chronic disease. However, as a person ages, the risk of chronic infection decreases such that between 20%-50% of children and less than 10% of older children or adults will progress from acute to chronic infection. Chronic HBV infections are the primary treatment goal for embodiments of the present invention, although ASO compositions of the present invention are also capable of treating HBV-related conditions, such as inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, and more.

“Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Comply” means the adherence with a recommended therapy by an individual.

“Comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Cure” means a method or course that restores health or a prescribed treatment for an illness.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

“Designing” or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected, the diluent may be a liquid, e.g. saline solution.

“Dosage unit” means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art.

“Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In other embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week or month.

“Dosing regimen” is a combination of doses designed to achieve one or more desired effects.

“Duration” means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent are administered.

“Effective amount” in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of active ingredient to a subject in need of such modulation, treatment or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount will vary depending upon the health and physical condition of the subject to be treated, the taxonomic group of subjects to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

“Fully modified motif” refers to an antisense compound comprising a contiguous sequence of nucleosides wherein essentially each nucleoside is a sugar modified nucleoside having uniform modification.

“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”

“Gap-widened” means an antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleotides positioned between 5′ and 3′ wing segments having from one to six nucleotides having modified sugar moieties.

“HBV” means mammalian hepatitis B virus, including human hepatitis B virus. The term encompasses geographical genotypes of hepatitis B virus, particularly human hepatitis B virus, as well as variant strains of geographical genotypes of hepatitis B virus.

“HBV antigen” means any hepatitis B virus antigen or protein, including core proteins such as “hepatitis B core antigen” or “HBcAG” and “hepatitis B E antigen” or “HBeAG” and envelope proteins such as “HBV surface antigen”, or “HBsAg” or “HBsAG”.

“Hepatitis B E antigen” or “HBeAg” or “HBeAG” is a secreted, non-particulate form of HBV core protein. HBV antigens HBeAg and HBcAg share primary amino acid sequences, so show cross-reactivity at the T cell level. HBeAg is not required for viral assembly or replication, although studies suggest they may be required for establishment of chronic infection. Neonatal infection with HBeAg-negative mutant often results in fulminant acute rather than chronic HBV infection (Terezawa et al (1991) Pediatr. Res. 29:5), whereas infection of young woodchucks with WHeAg-negative mutant results in a much lower rate of chronic WHV infection (Cote et al (2000) Hepatology 31:190). HBeAg may possibly function as a toleragen by inactivating core specific T cells through deletion or clonal anergy (Milich et al (1998) J. Immunol. 160:8102). There is a positive correlation between reduction of HBV viral load and antigens, and a decrease of expression, by T cells, of the inhibitory receptor programmed death-1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, upon antiviral therapy and HBeAg seroconversion (Evans et al (2008) Hepatology 48:759).

“HBV mRNA” means any messenger RNA expressed by hepatitis B virus.

“HBV nucleic acid” or “HBV DNA” means any nucleic acid encoding HBV. For example, in certain embodiments, a HBV nucleic acid includes, without limitation, any viral DNA sequence encoding a HBV genome or portion thereof, any RNA sequence transcribed from a viral DNA including any mRNA sequence encoding a HBV protein.

“HBV protein” means any protein secreted by hepatitis B virus The term encompasses various HBV antigens, including core proteins such as “Hepatitis E antigen”, “HBeAg” or “HBeAG” and envelope proteins such as “HBV surface antigen”, or “HBsAg”.

“HBV surface antigen”, or “HBsAg”, or “HBsAG” is the envelope protein of infectious HBV viral particles but is also secreted as a non-infectious particle with serum levels 1000-fold higher than HBV viral particles. The serum levels of HBsAg in an infected person or animal can be as high as 1000 μg/mL (Kann and Gehrlich (1998) Topley & Wilson's Microbiology and Microbial Infections, 9^(th) ed. 745). In acute HBV infections, the half-life of HBsAg in the serum, or serum t_(1/2), is 8.3 days (Chulanov et al (2003) J. Med. Virol. 69: 313). Internalization of HBsAg by myeloid dendritic cells inhibits up-regulation of co-stimulatory molecules (i.e. B7) and inhibits T cell stimulatory capacity (den Brouw et al (2008) Immunology 126:280), and dendritic cells from chronically infected patients also show deficits in expression of co-stimulatory molecules, secretion of IL-12, and stimulation of T cells in the presence of HBsAg (Zheng et al (2004) J. Viral Hepatitis 11:217). HBsAg specific CD8 cells from CHB patients show altered tetramer binding. These CD8 cells are not anergic but may have TCR topology that confers partial tolerance or ignorance (Reignat et al (2002) J. Exp. Med. 195:1089). Moreover, reduction in serum HBsAg >1 log at week 24 has a high predictive value (92%) for sustained virological response (SVR—defined as nondetectable HBV DNA by PCR at 1 year after treatment) during Peg-IFNα2a therapy (Moucari et al (2009) Hepatology 49:1151).

“Hepatitis B-related condition” or “HBV-related condition” means any disease, biological condition, medical condition, or event which is exacerbated, caused by, related to, associated with, or traceable to a hepatitis B infection, exposure, or illness. The term hepatitis B-related condition includes chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, liver disease related to transplantation, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

“Identifying an animal having an HBV infection” means identifying an animal having been diagnosed with an HBV; or, identifying an animal having any symptom of an HBV infection including, but not limited to chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, liver disease related to transplantation, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Individual compliance” means adherence to a recommended or prescribed therapy by an individual.

“Induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease” or the like, generally denote quantitative differences between two states. Such terms may refer to a statistically significant difference between the two states. For example, “an amount effective to inhibit the activity or expression of HBV” means that the level of activity or expression of HBV in a treated sample will quantitatively differ, and may be statistically significant, from the level of HBV activity or expression in untreated cells. Such terms are applied to, for example, levels of expression, and levels of activity.

“Inhibiting HBV” means reducing the level or expression of an HBV mRNA, DNA and/or protein. In certain embodiments, HBV is inhibited in the presence of an antisense compound targeting HBV, including an antisense oligonucleotide targeting HBV, as compared to expression of HBV mRNA, DNA and/or protein levels in the absence of a HBV antisense compound, such as an antisense oligonucleotide.

“Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

“Injection site reaction” means inflammation or abnormal redness of skin at a site of injection in an individual.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Intraperitoneal administration” means administration through infusion or injection into the peritoneum.

“Intravenous administration” means administration into a vein.

“Lengthened” antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.

“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

“Locked nucleic acid” or “LNA” or “LNA nucleosides” means nucleic acid monomers having a bridge connecting two carbon atoms between the 4′ and 2′ position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy (4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, as depicted below.

As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R₁)(R₂)]_(n)—, —C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—, —S(═O)_(x)— and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R₁ and R₂ is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: —[C(R₁)(R₂)]_(n)—, —[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—. Furthermore, other bridging groups encompassed with the definition of LNA are 4′-CH₂-2′, 4′-(CH₂)₂-2′,4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′-bridges, wherein each R₁ and R₂ is, independently, H, a protecting group or C₁-C₁₂ alkyl.

Also included within the definition of LNA according to the invention are LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring is connected to the 4′ carbon atom of the sugar ring, thereby forming a methyleneoxy (4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (—CH₂—) group connecting the 2′ oxygen atom and the 4′ carbon atom, for which the term methyleneoxy (4′-CH₂—O-2′) LNA is used. Furthermore; in the case of the bicylic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4′-CH₂CH₂—O-2′) LNA is used. α-L-methyleneoxy (4′-CH₂—O-2′), an isomer of methyleneoxy (4′-CH₂—O-2′) LNA is also encompassed within the definition of LNA, as used herein.

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

“Modified sugar” means substitution and/or any change from a natural sugar moiety.

“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

“Oligomeric compound” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, “peptide” refers to polypeptides and proteins.

“Pharmaceutically acceptable carrier” means a medium or diluent that does not interfere with the structure of the oligonucleotide. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject.

“Pharmaceutically acceptable derivative” encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to HBV is a pharmaceutical agent.

“Pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.

“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

“Prevention” or “preventing” refers to delaying or forestalling the onset or development of a condition or disease for a period of time from hours to days, preferably weeks to months.

“Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.

“Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.

“Recommended therapy” means a therapeutic regimen recommended by a medical professional for the treatment, amelioration, or prevention of a disease.

“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

“Salts” mean a physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

“Seroconversion” is defined as serum HBeAg absence plus serum HBeAb presence, if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence, if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.

“Shortened” or “truncated” versions of antisense oligonucleotides taught herein have one, two or more nucleosides deleted.

“Side effects” means physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

“Significant,” as used herein means measurable or observable, e.g, a significant result, such as, a significant improvement or significant reduction generally refers to a measurable or observable result, such as a measurable or observable improvement or reduction.

“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.

“Slows progression” means decrease in the development of the said disease.

“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

“Statistically Significant,” as used herein means a measurable or observable parameter that is unlikely to occur by chance.

“Subcutaneous administration” means administration just below the skin.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.

“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

“Treatment” refers to administering a composition to effect an alteration or improvement of the disease or condition.

“Unmodified” nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

“Validated target segment” is defined as at least an 8-nucleobase portion (i.e. 8 consecutive nucleobases) of a target region to which an active oligomeric compound is targeted.

“Wing segment” means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

CERTAIN EMBODIMENTS

Certain embodiments provide methods, compounds, and compositions for inhibiting HBV mRNA expression.

Certain embodiments provide antisense compounds targeted to a HBV nucleic acid. In certain embodiments, the HBV nucleic acid is the sequences set forth in GENBANK Accession No. U95551.1 (incorporated herein as SEQ ID NO: 1).

In certain embodiments, the compounds provided herein are or comprise a modified oligonucleotide. In certain embodiments the compounds comprise a modified oligonucleotide and a conjugate as described herein. In certain embodiments, the modified oligonucleotide is a pharmaceutically acceptable derivative.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. The HBV target can have a sequence recited in SEQ ID NO: 1 or a portion thereof or a variant thereof.

In certain embodiments, the compounds or modified oligonucleotides provided herein are 10 to 30 linked nucleosides in length and are targeted to HBV. In certain embodiments, the HBV target has the sequence recited in SEQ ID NO: 1. In certain embodiments, such compounds or oligonucleotides target one of the following nucleotide regions of HBV: CCTGCTGGTGGCTCCAGTTC (SEQ ID NO: 1273); AGAGTCTAGACTCGTGGTGGACTTCTCTCA (SEQ ID NO: 1354); CATCCTGCTGCTATGCCTCATCTTCTT (SEQ ID NO: 1276); CAAGGTATGTTGCCCGT (SEQ ID NO: 1277); CCTATGGGAGTGGGCCTCAG (SEQ ID NO: 1279; TGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCG (SEQ ID NO: 1287); TATATGGATGATGTGGT (SEQ ID NO:1359); TGCCAAGTGTTTGCTGA (SEQ ID NO:1360); TGCCGATCCATACTGCGGAACTCCT (SEQ ID NO: 1361); CCGTGTGCACTTCGCTTCACCTCTGCACGT (SEQ ID NO:1352); GGAGGCTGTAGGCATAAATTGGT (SEQ ID NO:1353); CTTTTTCACCTCTGCCTA (SEQ ID NO:1362); TTCAAGCCTCCAAGCTGTGCCTTGG (SEQ ID NO:1363); AGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGG (SEQ ID NO: 1274); TGGATGTGTCTGCGGCGTTTTATCAT (SEQ ID NO: 1275); TGTATTCCCATCCCATC (SEQ ID NO: 1278); TGGCTCAGTTTACTAGTGC (SEQ ID NO: 1280); GGGCTTTCCCCCACTGT (SEQ ID NO: 1281); TCCTCTGCCGATCCATACTGCGGAACTCCT (SEQ ID NO: 1282); CGCACCTCTCTTTACGCGG (SEQ ID NO: 1283); GGAGTGTGGATTCGCAC (SEQ ID NO: 1284); or GAAGAAGAACTCCCTCGCCT (SEQ ID NO: 1285). In certain embodiments, such compounds or oligonucleotides have a gap segment of 9, 10, or more linked deoxynucleosides. In certain embodiments, such compounds or oligonucleotides have a gap segment of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1, 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified nucleoside is a constrained ethyl (cEt) nucleoside.

In certain embodiments, the compounds or compositions comprise modified oligonucleotides consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising a portion at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleobases complementary to an equal length portion of any of the nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1. In certain embodiments, such oligonucleotides have a gap segment of 9, 10, or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1, 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified nucleoside is a constrained ethyl (cEt) nucleoside. In certain embodiments, each modified nucleoside in each wing segment is independently a 2′-MOE nucleoside or a nucleoside with a bicyclic sugar modification such as a constrained ethyl (cEt) nucleoside or LNA nucleoside.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, or 1379. In certain embodiments, such oligonucleotides have a gap segment of 9, 10, or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1, 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified nucleoside is a constrained ethyl (cEt) nucleoside.

In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, or 4 sugar modified nucleosides. In certain embodiments, each sugar modified nucleoside is independently a 2′-MOE nucleoside or a nucleoside with a bicyclic sugar moiety such as a constrained ethyl (cEt) nucleoside or LNA nucleoside. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, each sugar modified nucleoside is independently a 2′-MOE nucleoside or a bicyclic nucleoside such as a constrained ethyl (cEt) nucleoside or LNA nucleoside.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, or 1379. In certain embodiments, such oligonucleotides have a gap segment of 10 or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 2, 3, 4, 5, 6, 7, or 8 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272. In certain embodiments, such oligonucleotides have a gap segment of 10 or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified nucleoside is an LNA nucleoside. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of any of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538. In certain embodiments, such oligonucleotides have a gap segment of 10 or more linked deoxynucleosides. In certain embodiments, such gap segment is between two wing segments that independently have 1-5, 1-4, 1-3, 2-5, 2-4 or 2-3 linked modified nucleosides. In certain embodiments, one or more modified nucleosides in the wing segment have a modified sugar. In certain embodiments, the modified nucleoside is a 2′-substituted nucleoside. In certain embodiments, the modified nucleoside is a 2′-MOE nucleoside. In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides.

In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides, such as 2′-MOE nucleosides.

In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides, such as 2′-MOE nucleosides.

In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides.

In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides, such as 2′-MOE nucleosides.

In certain embodiments, the compounds or compositions comprise a salt of the modified oligonucleotide.

In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to SEQ ID NO: 1, as measured over the entirety of the modified oligonucleotide.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to any one of SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, as measured over the entirety of the modified oligonucleotide.

In certain embodiments, the compound or modified oligonucleotide is single-stranded.

In certain embodiments, the modified oligonucleotide consists of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides.

In certain embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In certain embodiments, at least one modified sugar comprises a 2′-O-methoxyethyl group (2′-O(CH₂)₂—OCH₃). In certain embodiments, the modified sugar comprises a 2′-O—CH₃ group.

In certain embodiments, at least one modified sugar is a bicyclic sugar. In certain embodiments, at least one modified sugar the bicyclic sugar comprises a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or 2. In certain embodiments, the bicyclic sugar comprises a 4′-CH₂—O-2′ bridge. In certain embodiments, the bicyclic sugar comprises a 4′-CH(CH₃)—O-2′ bridge.

In certain embodiments, at least one nucleoside of said modified oligonucleotide comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of a single-stranded modified oligonucleotide.

In certain embodiments, the modified oligonucleotide comprises: a) a gap segment consisting of linked deoxynucleosides; b) a 5′ wing segment consisting of linked nucleosides; and c) a 3′ wing segment consisting of linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment and each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and/or a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, each of the three linked nucleosides of the 5′ wing segment is a 2′-O-methoxyethyl sugar and each of the three linked nucleosides of the 3′ wing segment is a constrained ethyl (cEt) sugar. In other aspects, the three linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction. In other aspects, the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, the two linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of two linked nucleosides; the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the two linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of five linked nucleosides; the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of five linked nucleosides; each of the three linked nucleosides of the 5′ wing segment is a constrained ethyl (cEt) sugar; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the four linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar, constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 9 linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 8 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 7 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides; the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the five linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 7 linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of 7 linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of four linked nucleosides; the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of 10 linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine, wherein the five linked nucleosides of the 5′ wing are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and each of the five linked nucleosides of the 3′ wing are a 2′-O-methoxyethyl sugar.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. In some aspects, the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; each of the three linked nucleosides of the 5′ wing segment is a 2′-O-methoxyethyl sugar and each of the three linked nucleosides of the 3′ wing segment is a constrained ethyl (cEt) sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine residue is a 5-methylcytosine. In other aspects, the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; the three linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine residue is a 5-methylcytosine. In other aspects, the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the three linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. In some aspects, the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; the two linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, 2′-O-methoxyethyl sugar, constrained ethyl (cEt) sugar, and 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the two linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, wherein the three linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, wherein each of the three linked nucleosides of the 5′ wing segment is a constrained ethyl (cEt) sugar; each of the five linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar and a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine. In some aspects, the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and the four linked nucleosides of the 3′ wing segment are a 2′-O-methoxyethyl sugar, constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the four linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 9 linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) the 3′ wing segment consisting of four linked nucleosides, wherein the four linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 8 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 7 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, wherein the five linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; the five linked nucleosides of the 3′ wing segment are a constrained ethyl (cEt) sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, and a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 7 linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 7 linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides, wherein the six linked nucleosides of the 5′ wing segment are a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction; each of the four linked nucleosides of the 3′ wing segment is a 2′-O-methoxyethyl sugar; each internucleoside linkage is a phosphorothioate linkage; and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 10 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a constrained ethyl (cEt) sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of 10 linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine, wherein the five linked nucleosides of the 5′ wing are a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, a constrained ethyl (cEt) sugar, a 2′-deoxynucleoside, and a constrained ethyl (cEt) sugar in the 5′ to 3′ direction, and each of the five linked nucleosides of the 3′ wing are a 2′-O-methoxyethyl sugar.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of eight linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of eight linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of seven linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of seven linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of six linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of six linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of six linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of five linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of five linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of four linked nucleosides, the 3′ wing segment consisting of three linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides, the gap segment consisting of nine linked deoxynucleosides, the 5′ wing segment consisting of three linked nucleosides, the 3′ wing segment consisting of four linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides, the gap segment consisting of ten linked deoxynucleosides, the 5′ wing segment consisting of two linked nucleosides, the 3′ wing segment consisting of two linked nucleosides, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of eight linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of eight linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of seven linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of seven linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of six linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 18 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of six linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of six linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 17 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of four linked nucleosides; and c) a 3′ wing segment consisting of three linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of nine linked deoxynucleosides; b) a 5′ wing segment consisting of three linked nucleosides; and c) a 3′ wing segment consisting of four linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the compounds or compositions comprise a modified oligonucleotide consisting of 14 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of any of nucleobases set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, wherein the nucleobase sequence is complementary to SEQ ID NO: 1 and wherein the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of two linked nucleosides; and c) a 3′ wing segment consisting of two linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, each internucleoside linkage is a phosphorothioate linkage and each cytosine residue is a 5-methylcytosine.

In certain embodiments, the provided methods, compounds, and compositions inhibit HBV mRNA expression and/or DNA levels and or protein levels and/or antigen levels.

Another embodiment provides a method for treating a HBV-related diseases, disorders, and conditions in a mammal, the method comprising administering a therapeutically effective amount of any pharmaceutical composition as described above to a mammal in need thereof, so as to treat the HBV-related diseases, disorders, and condition. In related embodiments, the mammal is a human and the HBV-related disease, disorder, and condition is a hepatitis B virus infection from a human hepatitis B virus. More particularly, the human hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).

In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 1:1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-274; 245-260, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-266, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-270, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-437, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-754, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 678-693, 679-694, 679-707, 679-698, 679-701, 679-702, 679-707, 680-695, 680-699, 679-699, 681-706, 681-696, 682-697, 682-706, 682-707, 682-702, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 738-753, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1176-1285, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1889, 1778-1794, 1779-1795, 1780-1799, 1785-1800, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1822-1837, 1823-1843, 1823-1838, 1824-1839, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1866-1881, 1867-1882, 1867-1886, 1868-1883, 1869-1885, 1869-1884, 1870-1885, 1871-1886, 1872-1887, 1874-1889, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.

In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the following nucleotide regions of SEQ ID NO: 1:1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-274; 245-260, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-266, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-270, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-437, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-754, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 678-693, 679-694, 679-707, 679-698, 679-701, 679-702, 679-707, 680-695, 680-699, 679-699, 681-706, 681-696, 682-697, 682-706, 682-707, 682-702, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 738-753, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1176-1285, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1889, 1778-1794, 1779-1795, 1780-1799, 1785-1800, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1822-1837, 1823-1843, 1823-1838, 1824-1839, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1866-1881, 1867-1882, 1867-1886, 1868-1883, 1869-1885, 1869-1884, 1870-1885, 1871-1886, 1872-1887, 1874-1889, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.

In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the HBV pre-S1 second portion gene region corresponding to nucleotide region 1-1932 of SEQ ID NO: 1. In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the HBV pre-S1 first portion gene region corresponding to nucleotide region 2831-3182 of SEQ ID NO: 1.

In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the HBV pre-S1 second portion gene region corresponding to nucleotide region 1-1932 of SEQ ID NO: 1. In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the HBV pre-S1 first portion gene region corresponding to nucleotide region 2831-3182 of SEQ ID NO: 1.

In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-274; 245-260, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-266, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-270, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-437, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-754, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 678-693, 679-694, 679-707, 679-698, 679-701, 679-702, 679-707, 680-695, 680-699, 679-699, 681-706, 681-696, 682-697, 682-706, 682-707, 682-702, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 738-753, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1176-1285, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1889, 1778-1794, 1779-1795, 1780-1799, 1785-1800, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1822-1837, 1823-1843, 1823-1838, 1824-1839, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1866-1881, 1867-1882, 1867-1886, 1868-1883, 1869-1885, 1869-1884, 1870-1885, 1871-1886, 1872-1887, 1874-1889, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.

In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 58-73, 58-74, 58-77, 59-74, 60-75, 61-76, 62-77, 245-274; 245-260, 250-265, 251-266, 252-267, 253-268, 254-269, 255-270, 256-271, 256-272, 258-273, 259-274, 380-399, 382-401, 411-437, 411-427, 411-426, 412-427, 413-428, 413-432, 414-429, 415-430, 416-431, 417-432, 418-433, 419-434, 420-435, 421-436, 422-437, 457-472, 458-473, 639-754, 639-658, 639-654, 641-656, 642-657, 643-658, 670-754, 670-706, 670-685, 671-686, 672-687, 673-688, 678-693, 679-694, 680-695, 681-706, 681-696, 682-697, 683-698, 684-699, 685-700, 686-701, 687-702, 688-703, 689-704, 690-705, 691-706, 738-754, 738-753, 739-754, 1176-1285, 1176-1191, 1177-1192, 1261-1285, 1261-1276, 1262-1277, 1263-1278, 1264-1279, 1265-1280, 1266-1281, 1267-1282, 1268-1283, 1269-1284, 1270-1285, 1577-1606, 1577-1592, 1578-1593, 1579-1594, 1580-1595, 1581-1596, 1582-1597, 1583-1598, 1584-1599, 1585-1600, 1586-1601, 1587-1602, 1588-1603, 1589-1604, 1590-1605, 1591-1606, 1778-1889, 1778-1800, 1778-1793, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1796, 1782-1797, 1783-1798, 1784-1799, 1785-1800, 1822-1839, 1822-1837, 1823-1838, 1824-1839, 1866-1881, 1867-1882, 1868-1883, 1869-1884, 1870-1885, 1871-1886, 1872-1887, or 1874-1889, and wherein at least one nucleoside of the compound or modified oligonucleotide comprises at least one 2′-O-methoxyethyl or constrained ethyl (cEt) sugar.

In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 1: 58-73, 58-74, 58-77, 59-74, 59-75, 60-75, 60-76, 61-76, 61-77, 62-77, 253-272, 253-269, 254-270, 255-271, 256-272, 411-437, 411-426, 411-427, 411-430, 412-427, 412-428, 412-431, 413-428, 413-429, 413-432, 414-429, 414-430, 414-433, 415-430, 415-431, 415-434, 416-431, 416-432, 416-435, 417-432, 417-433, 417-436, 418-433, 418-434, 418-437, 457-472, 457-473, 458-473, 670-706, 670-685, 670-686, 671-686, 671-687, 672-687, 672-688, 673-688, 687-702, 687-703, 687-706, 688-703, 688-704, 689-704, 689-705, 690-705, 690-706, 691-706, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1269-1284, 1269-1285, 1270-1285, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1794, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1799, and 1784-1800.

In certain embodiments, an antisense compound or oligonucleotide targeted to a HBV nucleic acid target the following nucleotide regions of SEQ ID NO: 1: 58-73, 58-74, 58-77, 59-74, 59-75, 60-75, 60-76, 61-76, 61-77, 62-77, 253-272, 253-269, 254-270, 255-271, 256-272, 411-437, 411-426, 411-427, 411-430, 412-427, 412-428, 412-431, 413-428, 413-429, 413-432, 414-429, 414-430, 414-433, 415-430, 415-431, 415-434, 416-431, 416-432, 416-435, 417-432, 417-433, 417-436, 418-433, 418-434, 418-437, 457-472, 457-473, 458-473, 670-706, 670-685, 670-686, 671-686, 671-687, 672-687, 672-688, 673-688, 687-702, 687-703, 687-706, 688-703, 688-704, 689-704, 689-705, 690-705, 690-706, 691-706, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1269-1284, 1269-1285, 1270-1285, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1794, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1799, and 1784-1800.

In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-73, 58-74, 58-77, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-75, 60-76, 60-79, 61-76, 61-77, 61-80, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-268, 251-269, 245-269, 245-266, 245-261, 250-266, 250-267, 250-268, 250-269, 251-270, 253-269, 253-272, 253-274, 254-270, 254-274, 255-271, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-275, 255-276, 256-272, 256-276, 253-275, 256-279, 257-276, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-426, 411-427, 411-430, 411-431, 411-437, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-429, 413-432, 413-433, 414-427, 415-427, 414-429, 414-430, 414-433, 415-428, 415-429, 415-430, 415-431, 415-434, 416-431, 416-432, 416-429, 416-435, 417-432, 417-433, 417-436, 418-433, 418-435, 418-434, 418-437, 419-435, 419-434, 420-435, 419-432, 419-434, 422-441, 423-436, 425-465, 454-473, 454-472, 457-476, 457-472, 457-473, 454-476, 455-472, 457-485, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 642-754, 653-672, 662-685, 665-685, 665-689, 668-687, 670-706, 670-685, 670-686, 670-689, 671-690, 671-691, 671-686, 671-687, 672-693, 672-697, 672-707, 672-687, 672-688, 673-688, 674-693, 679-707, 679-698, 679-701, 679-702, 679-707, 680-699, 679-699, 682-706, 682-707, 682-702, 682-701, 687-754, 688-704, 689-709, 689-710, 690-705, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-702, 687-703, 687-706, 688-703, 688-704, 688-705, 689-704, 689-705, 689-708, 690-705, 690-706, 690-709, 691-706, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-754, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 863-882, 863-885, 878-900, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 972-1015, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1245-1265, 1251-1280, 1262-1285, 1251-1285, 1259-1296, 1259-1290, 1259-1287, 1261-1296, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1296, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1605, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1571-1598, 1579-1594, 1579-1594, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1580-1605, 1580-1602, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1586-1652, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1799, 1780-1796, 1780-1795, 1781-1797, 1781-1796, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1782-1797, 1782-1798, 1783-1798, 1783-1799, 1784-1800, 1784-1799, 1779-1799, 1778-1794, 1779-1795, 1780-1799, 1794-1813, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1823-1843, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1867-1886, 1869-1885, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.

In certain embodiments, antisense compounds or oligonucleotides target a region of a HBV nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a HBV nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or oligonucleotide target the following nucleotide regions of SEQ ID NO: 1: 233-264, 242-263, 243-262, 244-263, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-268, 251-269, 245-269, 245-266, 245-261, 250-266, 250-267, 250-268, 250-269, 251-270, 253-272, 253-274, 254-274, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 256-275, 255-276, 256-276, 253-275, 256-279, 257-276, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 405-424, 409-428, 405-428, 411-430, 411-431, 411-431, 412-431, 411-426, 411-427, 412-428, 412-431, 412-427, 413-433, 413-432, 413-428, 413-433, 411-427, 414-427, 415-427, 415-428, 415-429, 416-432, 416-429, 418-435, 418-434, 419-435, 419-434, 420-435, 419-432, 419-434, 422-441, 423-436, 425-465, 584-606, 611-645, 617-363, 623-642, 617-645, 642-754, 653-672, and wherein at least one nucleoside of the compound or modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 50% inhibition: 1-20, 10-29, 10-56, 13-38, 13-35, 19-38, 25-47, 25-50, 25-56, 43-68, 43-63, 55-74, 58-77, 58-74, 58-73, 58-79, 58-80, 58-84, 59-74, 59-75, 59-80, 60-79, 60-75, 60-76, 61-80, 61-76, 61-77, 62-77, 63-84, 68-114, 101-123, 98-123, 113-138, 116-138, 131-150, 137-162, 152-186, 158-177, 167-186, 191-215, 196-224, 196-215, 196-218, 199-228, 199-218, 199-224, 200-224, 205-224, 206-228, 218-237, 224-243, 233-264, 242-263, 243-262, 244-263, 245-264, 246-266, 247-266, 247-269, 247-270, 245-267, 251-267, 245-266, 250-269, 251-268, 251-269, 245-269, 245-266, 245-261, 250-265, 250-266, 250-267, 250-268, 250-269, 251-266, 251-270, 252-267, 253-268, 253-269, 253-272, 253-274, 254-269, 254-270, 254-274, 255-274, 255-401, 255-400, 255-274, 255-273, 255-272, 255-271, 255-270, 256-271, 256-272, 256-275, 255-276, 256-276, 253-275, 256-279, 257-276, 258-273, 259-274, 260-279, 262-281, 262-321, 262-315, 262-312, 265-312, 266-288, 266-291, 266-285, 281-321, 281-303, 290-321, 290-312, 292-311, 290-312, 293-312, 293-315, 293-321, 296-321, 302-321, 324-343, 339-361, 339-367, 348-367, 342-367, 358-392, 358-378, 360-392, 360-383, 360-388, 360-385, 362-381, 366-388, 369-388, 366-385, 366-392, 370-389, 370-392, 380-399, 382-401, 384-433, 384-400, 384-401, 385-401, 405-424, 409-428, 405-428, 411-430, 411-431, 411-431, 412-431, 411-426, 411-427, 411-430, 411-437, 412-428, 412-431, 412-427, 413-432, 413-428, 413-429, 413-433, 411-427, 414-427, 414-429, 414-430, 414-433, 415-427, 415-428, 415-429, 415-430, 415-431, 415-434, 416-435, 416-432, 416-431, 416-429, 417-432, 417-433, 417-436, 418-437, 418-435, 418-434, 418-433, 419-435, 419-434, 420-435, 419-432, 419-434, 421-436, 422-441, 422-437, 423-436, 425-465, 454-473, 454-472, 457-476, 454-476, 455-472, 457-485, 457-473, 457-472, 458-485, 458-483, 458-477, 458-473, 459-485, 460-485, 463-498, 463-485, 466-485, 463-482, 457-491, 458-491, 459-491, 460-491, 463-491, 466-491, 472-491, 472-493, 473-492, 475-491, 459-494, 460-494, 463-494, 466-494, 467-498, 472-494, 475-494, 457-473, 457-472, 458-494, 454-494, 457-494, 457-473, 485-513, 470-493, 476-519, 485-519, 500-519, 512-534, 512-550, 524-546, 536-559, 548-567, 548-570, 550-570, 548-594, 554-573, 548-576, 560-594, 584-606, 611-645, 617-363, 623-642, 617-645, 639-654, 641-656, 642-657, 642-754, 643-658, 653-672, 662-685, 665-685, 665-689, 668-687, 670-689, 670-706, 670-685, 670-686, 671-686, 671-687, 671-690, 671-691, 672-687, 672-688, 672-693, 672-697, 672-707, 673-688, 674-693, 678-693, 679-707, 679-694, 679-698, 679-701, 679-702, 679-707, 680-699, 679-699, 680-695, 681-696, 682-706, 682-707, 682-702, 682-701, 682-697, 683-698, 684-699, 685-700, 686-701, 687-702, 687-703, 687-706, 687-754, 688-703, 688-704, 689-704, 689-705, 689-709, 689-710, 690-705, 690-706, 691-706, 679-705, 679-710, 679-706, 690-710, 691-710, 690-754, 690-706, 684-703, 687-705, 687-703, 687-706, 688-705, 689-708, 690-709, 692-711, 693-716, 693-712, 695-715, 697-716, 697-716, 690-716, 724-746, 724-752, 724-754, 724-758, 733-752, 738-753, 738-754, 739-758, 739-754, 739-775, 739-754, 740-754, 742-785, 742-773, 757-776, 757-785, 790-815, 793-812, 811-833, 811-844, 814-833, 811-906, 820-839, 822-844, 822-867, 823-842, 845-864, 845-867, 854-906, 845-909, 845-906, 854-876, 854-873, 863-882, 863-885, 878-900, 878-897, 887-906, 899-918, 899-933, 899-958, 905-927, 905-933, 914-933, 936-958, 936-955, 945-964, 951-970, 951-985, 951-1044, 951-1024, 951-1056, 951-997, 960-985, 963-1044, 963-1024, 963-997, 966-985, 972-1015, 978-997, 1025-1044, 1031-1056, 1037-1056, 1046-1083, 1049-1068, 1070-1089, 1070-1095, 1082-1101, 1081-1134, 1081-1143, 1082-1101, 1088-1107, 1088-1134, 1094-1119, 1097-1119, 1112-1134, 1118-1143, 1118-1146, 1088-1146, 1121-1140, 1127-1146, 1127-1193, 1150-1193, 1156-1187, 1165-1187, 1170-1192, 1171-1191, 1172-1191, 1176-1192, 1177-1192, 1176-1191, 1203-1297, 1206-1228, 1206-1255, 1209-1228, 1215-1255, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1265, 1251-1280, 1251-1285, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1296, 1259-1290, 1259-1287, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1261-1296, 1262-1277, 1262-1278, 1262-1281, 1262-1285, 1262-1296, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1264-1297, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1296, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1271-1290, 1271-1296, 1277-1296, 1261-1290, 1262-1290, 1268-1290, 1263-1305, 1259-1305, 1259-1305, 1266-1305, 1259-1302, 1275-1294, 1281-1306, 1281-1324, 1281-1336, 1782-1797, 1282-1301, 1286-1306, 1290-1324, 1293-1318, 1290-1324, 1293-1315, 1296-1315, 1311-1336, 1311-1333, 1326-1345, 1353-1381, 1359-1378, 1395-1414, 1498-1532, 1498-1523, 1498-1535, 1510-1529, 1515-1535, 1515-1563, 1515-1596, 1515-1605, 1515-1602, 1515-1540, 1515-1535, 1518-1605, 1518-1602, 1518-1537, 1521-1563, 1521-1540, 1550-1655, 1550-1563, 1550-1569, 1553-1578, 1553-1599, 1553-1590, 1565-1584, 1571-1595, 1577-1606, 1577-1605, 1577-1596, 1577-1592, 1577-1593, 1578-1593, 1578-1594, 1578-1597, 1578-1598, 1579-1594, 1579-1595, 1579-1598, 1571-1598, 1580-1605, 1580-1602, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1553-1655, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1586-1652, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1651-1673, 1655-1679, 1695-1720, 1716-1738, 1743-1763, 1743-1768, 1764-1783, 1773-1792, 1777-1796, 1777-1800, 1778-1800, 1778-1793, 1778-1794, 1778-1797, 1779-1798, 1779-1797, 1779-1796, 1779-1795, 1779-1794, 1780-1796, 1781-1797, 1781-1796, 1781-1800, 1781-1797, 1782-1799, 1784-1800, 1779-1799, 1778-1794, 1779-1795, 1780-1799, 1794-1813, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1798, 1783-1799, 1784-1799, 1784-1800, 1785-1800, 1806-1837, 1806-1828, 1806-1825, 1809-1828, 1812-1843, 1812-1837, 1812-1831, 1815-1843, 1815-1844, 1815-1840, 1815-1834, 1818-1837, 1821-1840, 1821-1844, 1821-1837, 1822-1843, 1822-1839, 1823-1843, 1827-1846, 1861-1884, 1861-1880, 1865-1885, 1867-1886, 1869-1885, 1876-1895, 1888-1914, 1888-1908, 1891-1910, 1891-1914, 1895-1938, 1895-1935, 1913-1935, 1898-1920, 1907-1929, 1913-1935, 1918-1934, 1919-1938, 1919-1934, 1921-1934, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2393, 2381-2397, 2368-2394, 2379-2394, 2381-2396, 2368-2397, 2368-2396, 2420-2439, 2458-2476, 2459-2478, 2819-2838, 2818-2838, 2873-2892, and 3161-3182.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 60% inhibition: 1-20, 10-29, 10-53, 13-38, 25-50, 43-68, 55-74, 58-84, 58-77, 58-74, 58-73, 58-79, 59-80, 59-74, 59-75, 60-75, 60-76, 61-77, 61-76, 61-80, 62-77, 68-114, 98-123, 101-123, 113-138, 116-138, 131-150, 137-162, 152-186, 191-215, 196-224, 196-215, 199-228, 199-218, 200-223, 199-218, 205-224, 206-228, 218-237, 224-243, 233-263, 244-263, 245-264, 247-266, 250-265, 251-266, 252-267, 253-272, 253-269, 251-267, 253-274, 254-270, 255-276, 256-279, 256-276, 256-274, 256-272, 256-271, 258-273, 259-274, 265-388, 265-284, 266-291, 266-288, 260-279, 281-321, 281-303, 290-321, 290-312, 293-312, 296-315, 302-321, 324-343, 339-367, 339-361, 342-367, 348-367, 358-392, 358-378, 360-392, 360-379, 366-392, 366-385, 369-388, 370-392, 382-401, 405-428, 405-424, 409-428, 411-436, 411-433, 411-431, 411-426, 411-430, 411-427, 412-431, 412-428, 412-427, 413-428, 413-429, 413-433, 414-433, 414-430, 414-429, 414-433, 415-430, 415-431, 415-434, 415-435, 415-436, 416-429, 416-434, 416-431, 416-432, 416-436, 416-435, 417-436, 417-433, 417-432, 418-434, 418-433, 418-437, 419-434, 420-435, 421-436, 422-437, 423-436, 425-465, 454-472, 455-472, 457-476, 457-472, 457-473, 458-485, 458-473, 458-483, 463-498, 467-498, 463-482, 470-493, 472-491, 485-519, 485-513, 500-519, 512-534, 524-546, 536-558, 548-567, 554-573, 548-576, 560-594, 584-606, 608-648, 639-654, 640-656, 641-656, 642-657, 642-658, 643-658, 653-672, 662-685, 665-685, 670-706, 670-689, 670-685, 670-686, 671-690, 671-686, 671-687, 672-707, 672-697, 672-693, 672-687, 672-688, 673-688, 679-707, 679-698, 679-694, 680-695, 681-696, 682-697, 682-701, 683-698, 684-699, 685-700, 686-701, 687-754, 687-702, 687-705, 687-703, 687-706, 688-704, 688-703, 688-704, 688-705, 688-707, 689-710, 689-709, 689-705, 689-704, 690-754, 690-705, 690-706, 691-706, 691-710, 692-711, 697-716, 724-758, 724-754, 724-752, 724-746, 738-754, 738-753, 739-754, 742-785, 757-785, 790-815, 811-906, 811-844, 811-833, 822-867, 822-844, 823-842, 845-867, 854-906, 854-873, 878-897, 899-958, 899-933, 936-958, 945-964, 951-1044, 951-1024, 951-985, 951-997, 963-1044, 963-1024, 963-997, 966-985, 978-997, 1031-1056, 1046-1083, 1070-1095, 1081-1143, 1081-1134, 1082-1101, 1088-1146, 1088-1134, 1118-1146, 1118-1143, 1127-1193, 1170-1189, 1176-1192, 1176-1191, 1177-1192, 1203-1297, 1206-1255, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1251-1270, 1251-1285, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1276, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1281, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1279, 1263-1282, 1264-1297, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1281-1336, 1281-1324, 1281-1306, 1286-1305, 1290-1324, 1311-1336, 1326-1345, 1353-1381, 1395-1414, 1498-1535, 1498-1532, 1515-1535, 1515-1534, 1521-1540, 1550-1655, 1553-1599, 1553-1590, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1716-1738, 1743-1763, 1764-1783, 1773-1792, 1777-1800, 1777-1797, 1655-1674, 1778-1794, 1778-1800, 1781-1800, 1781-1797, 1784-1800, 1779-1799, 1778-1794, 1778-1797, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1794-1813, 1806-1837, 1806-1825, 1812-1837, 1812-1831, 1815-1844, 1815-1834, 1818-1837, 1821-1837, 1822-1838, 1827-1846, 1861-1884, 1821-1840, 1866-1885, 1867-1886, 1888-1914, 1888-1907, 1891-1914, 1895-1938, 1895-1935, 1919-1938, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2397, 2368-2396, 2368-2394, 2368-2393, 2379-2394, 2381-2396, 2420-2439, 2458-2476, 2819-2838, 2873-2892, and 3161-3182.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 65% inhibition: 1-20, 10-29, 10-53, 13-38, 25-50, 43-68, 55-74, 58-84, 58-79, 58-74, 58-73, 58-77, 59-75, 59-80, 58-77, 60-75, 60-76, 61-77, 61-76, 61-80, 62-77, 68-114, 98-123, 101-123, 113-138, 116-138, 131-150, 137-162, 152-186, 191-215, 196-215, 199-228, 199-218, 200-223, 199-218, 205-224, 206-228, 218-237, 224-243, 233-263, 244-263, 245-264, 250-265, 251-266, 253-269, 253-274, 255-276, 256-279, 256-276, 256-274, 256-272, 256-271, 247-266, 253-272, 258-273, 266-291, 266-288, 260-279, 281-321, 281-303, 290-321, 290-312, 296-315, 293-312, 302-321, 324-343, 339-367, 339-361, 342-367, 348-367, 358-392, 358-378, 360-392, 360-379, 366-392, 366-385, 369-388, 370-392, 382-401, 405-428, 405-424, 409-428, 411-433, 411-431, 411-430, 411-427, 411-426, 412-431, 412-428, 412-427, 413-433, 413-428, 413-429, 413-432, 414-433, 414-430, 414-429, 415-430, 415-431, 415-434, 415-435, 415-436, 416-434, 416-436, 416-435, 416-432, 416-431, 417-436, 417-433, 417-432, 418-433, 418-434, 418-437, 420-435, 422-437, 423-436, 425-465, 454-472, 455-472, 457-472, 458-485, 458-483, 458-473, 463-498, 467-498, 457-476, 470-493, 472-491, 485-519, 485-513, 500-519, 512-534, 524-546, 536-558, 548-567, 554-573, 548-576, 560-594, 584-606, 608-648, 639-654, 640-656, 641-656, 642-657, 642-658, 643-658, 653-672, 662-685, 665-685, 670-685, 670-706, 670-689, 670-686, 670-685, 671-686, 671-687, 671-690, 672-688, 672-687, 672-707, 672-697, 672-693, 673-688, 679-698, 680-695, 681-696, 682-697, 682-701, 683-698, 684-699, 685-700, 686-701, 687-702, 688-703, 688-707, 687-754, 690-754, 690-706, 690-705, 687-705, 687-703, 687-706, 687-702, 688-705, 688-703, 688-704, 689-705, 691-706, 692-711, 697-716, 724-758, 724-754, 724-752, 724-746, 738-754, 739-754, 742-785, 757-785, 790-815, 811-906, 811-844, 811-833, 822-867, 822-844, 823-842, 845-867, 854-906, 854-873, 878-897, 899-958, 899-933, 936-958, 945-964, 951-1044, 951-1024, 951-985, 951-997, 963-1044, 963-1024, 963-997, 966-985, 978-997, 1031-1056, 1046-1083, 1070-1095, 1081-1143, 1081-1134, 1082-1101, 1088-1146, 1088-1134, 1118-1146, 1118-1143, 1127-1193, 1170-1189, 1176-1192, 1177-1192, 1203-1297, 1206-1255, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1251-1270, 1251-1285, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1281, 1262-1277, 1262-1278, 1263-1278, 1263-1279, 1263-1282, 1264-1297, 1264-1279, 1264-1280, 1264-1283, 1265-1280, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1281-1336, 1281-1324, 1281-1306, 1290-1324, 1311-1336, 1326-1345, 1353-1381, 1395-1414, 1498-1535, 1498-1532, 1515-1535, 1515-1534, 1550-1655, 1553-1599, 1553-1590, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1655-1674, 1716-1738, 1743-1763, 1764-1783, 1773-1792, 1777-1800, 1777-1797, 1778-1800, 1778-1797, 1779-1799, 1778-1794, 1779-1794, 1779-1795, 1779-1798, 1780-1796, 1780-1799, 1780-1795, 1781-1796, 1781-1797, 1781-1800, 1782-1797, 1794-1813, 1806-1837, 1806-1825, 1812-1837, 1812-1831, 1815-1844, 1815-1834, 1818-1837, 1821-1837, 1822-1838, 1827-1846, 1861-1884, 1866-1885, 1867-1886, 1888-1914, 1888-1907, 1891-1914, 1895-1938, 1895-1935, 1919-1938, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2397, 2368-2396, 2368-2394, 2368-2393, 2379-2394, 2381-2396, 2420-2439, 2458-2476, 2819-2838, 2873-2892, and 3161-3182.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 70% inhibition: 1-20, 10-29, 10-53, 13-38, 25-50, 43-68, 55-74, 58-84, 58-79, 58-74, 59-75, 59-80, 58-77, 60-75, 60-76, 61-77, 68-114, 98-123, 101-123, 113-138, 116-138, 131-150, 137-162, 152-186, 191-215, 199-228, 199-218, 200-223, 205-224, 206-228, 218-237, 224-243, 233-263, 244-263, 245-264, 253-269, 253-274, 255-276, 256-279, 256-276, 256-274, 256-272, 247-266, 250-265, 251-266, 253-272, 256-271, 266-291, 266-288, 260-279, 281-321, 281-303, 290-321, 290-312, 293-312, 302-321, 324-343, 339-367, 339-361, 342-367, 348-367, 358-392, 358-378, 360-392, 360-379, 366-392, 366-385, 370-392, 382-401, 405-428, 405-424, 409-428, 411-433, 411-431, 411-430, 411-427, 411-426, 412-431, 412-428, 412-427, 413-428, 413-429, 413-432, 414-433, 414-430, 414-429, 415-430, 414-433, 415-434, 415-435, 415-436, 416-431, 416-434, 416-436, 416-435, 416-432, 417-436, 417-433, 418-433, 418-437, 423-436, 425-465, 454-472, 455-472, 457-472, 457-476, 458-473, 458-485, 458-483, 463-498, 467-498, 457-476, 470-493, 470-493, 472-491, 485-519, 485-513, 485-519, 485-513, 500-519, 512-534, 524-546, 536-558, 548-567, 554-573, 548-576, 560-594, 584-606, 608-648, 639-654, 640-656, 641-656, 642-657, 642-658, 643-658, 653-672, 662-685, 665-685, 670-706, 670-689, 670-685, 670-686, 671-690, 671-686, 671-687, 672-687, 672-688, 672-707, 672-697, 672-693, 673-688, 679-698, 681-696, 682-697, 682-701, 683-698, 684-699, 686-701, 687-702, 687-754, 687-702, 688-703, 690-754, 690-706, 687-705, 687-703, 687-706, 692-711, 697-716, 724-758, 724-754, 724-752, 724-746, 738-754, 739-754, 738-754, 742-785, 757-785, 790-815, 811-906, 811-844, 811-833, 822-867, 822-844, 845-867, 854-906, 854-873, 878-897, 899-958, 899-933, 936-958, 945-964, 951-1044, 951-1024, 951-985, 951-997, 963-1044, 963-1024, 963-997, 966-985, 978-997, 1031-1056, 1046-1083, 1070-1095, 1081-1143, 1081-1134, 1082-1101, 1088-1146, 1088-1134, 1118-1146, 1118-1143, 1127-1193, 1170-1189, 1176-1192, 1177-1192, 1203-1297, 1206-1255, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1251-1285, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1281, 1262-1277, 1262-1278, 1263-1278, 1263-1279, 1263-1282, 1264-1297, 1264-1279, 1264-1280, 1264-1283, 1265-1281, 1265-1284, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1281-1336, 1281-1324, 1281-1306, 1290-1324, 1311-1336, 1326-1345, 1353-1381, 1395-1414, 1498-1535, 1498-1532, 1515-1535, 1550-1655, 1553-1599, 1553-1590, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1602, 1586-1605, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1642-1664, 1651-1720, 1716-1738, 1743-1763, 1764-1783, 1773-1792, 1777-1800, 1777-1797, 1778-1800, 1778-1797, 1779-1799, 1778-1794, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1800, 1782-1797, 1794-1813, 1806-1837, 1806-1825, 1812-1837, 1812-1831, 1815-1844, 1815-1834, 1818-1837, 1821-1837, 1822-1838, 1827-1846, 1861-1884, 1866-1885, 1867-1886, 1888-1914, 1888-1907, 1891-1914, 1895-1938, 1895-1935, 1919-1938, 1928-1956, 1957-1976, 2035-2057, 2083-2141, 2230-2261, 2278-2297, 2281-2300, 2284-2303, 2368-2397, 2368-2396, 2368-2394, 2368-2393, 2379-2394, 2381-2396, 2420-2439, 2458-2476, 2819-2838, 2873-2892, and 3161-3182.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 75% inhibition: 13-32, 16-35, 19-38, 25-44, 28-47, 31-50, 43-62, 46-65, 49-68, 55-74, 58-82, 58-74, 58-77, 59-75, 60-75, 60-76, 61-77, 65-84, 98-117, 101-120, 104-123, 116-135, 119-138, 131-150, 137-156, 140-159, 143-162, 158-177, 161-180, 164-183, 167-186, 200-219, 203-226, 209-228, 218-237, 233-252, 236-255, 239-258, 242-264, 247-266, 251-266, 253-272, 255-276, 266-285, 269-288, 281-300, 284-303, 290-313, 298-317, 302-321, 324-343, 339-358, 342-361, 348-367, 358-381, 364-383, 366-386, 370-389, 373-392, 382-401, 405-424, 409-428, 411-430, 411-426, 411-427, 412-427, 412-431, 413-428, 413-429, 413-432, 414-436, 414-430, 414-429, 415-430, 416-431, 416-432, 417-433, 418-437, 422-441, 425-444, 428-447, 434-453, 440-459, 443-462, 446-465, 456-477, 458-473, 464-483, 470-493, 476-495, 479-498, 488-507, 491-510, 494-513, 500-519, 512-531, 515-534, 524-543, 527-546, 536-555, 539-558, 560-579, 566-585, 569-588, 572-591, 575-594, 584-603, 587-606, 608-627, 614-633, 617-636, 620-639, 623-642, 626-645, 629-648, 639-654, 641-656, 642-657, 643-658, 653-672, 665-684, 668-688, 670-706, 670-686, 670-685, 671-691, 671-687, 671-686, 672-688, 673-688, 679-703, 681-696, 682-697, 686-701, 686-706, 687-702, 687-703, 688-703, 689-708, 693-712, 695-714, 696-715, 697-716, 727-746, 739-754, 742-761, 748-767, 751-770, 754-773, 757-776, 760-779, 763-782, 766-785, 790-809, 793-812, 796-815, 811-830, 814-833, 817-836, 820-839, 822-844, 845-864, 854-873, 857-876, 863-882, 866-885, 872-891, 875-894, 878-897, 881-900, 884-903, 887-906, 899-918, 902-921, 905-924, 908-927, 911-930, 914-933, 936-955, 939-958, 951-970, 954-973, 957-976, 960-979, 963-982, 966-985, 969-988, 972-991, 975-994, 978-997, 996-1015, 1002-1021, 1025-1044, 1031-1050, 1034-1053, 1037-1056, 1046-1065, 1049-1068, 1052-1071, 1055-1074, 1058-1077, 1061-1080, 1064-1083, 1070-1089, 1073-1092, 1076-1095, 1082-1101, 1088-1107, 1094-1113, 1097-1116, 1100-1119, 1103-1122, 1106-1125, 1109-1128, 1112-1131, 1115-1134, 1121-1140, 1127-1146, 1153-1172, 1156-1175, 1159-1178, 1162-1181, 1165-1184, 1168-1191, 1174-1193, 1206-1225, 1209-1228, 1212-1231, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1239-1258, 1242-1261, 1245-1264, 1251-1270, 1254-1273, 1254-1279, 1257-1283, 1257-1276, 1258-1277, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1263-1278, 1263-1279, 1263-1282, 1264-1279, 1264-1280, 1264-1283, 1265-1281, 1265-1284, 1266-1281, 1266-1282, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1268-1287, 1269-1284, 1269-1285, 1270-1285, 1272-1291, 1275-1294, 1282-1303, 1286-1306, 1290-1309, 1293-1312, 1296-1315, 1299-1318, 1305-1324, 1311-1330, 1314-1333, 1317-1336, 1353-1381, 1356-1375, 1359-1378, 1498-1517, 1501-1520, 1504-1523, 1510-1529, 1553-1572, 1556-1575, 1559-1578, 1562-1581, 1565-1584, 1571-1590, 1574-1599, 1577-1606, 1577-1592, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1594, 1579-1595, 1579-1598, 1580-1595, 1580-1596, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1598, 1582-1601, 1582-1602, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1600, 1584-1603, 1585-1600, 1585-1601, 1585-1604, 1586-1601, 1586-1605, 1586-1602, 1587-1602, 1587-1603, 1587-1606, 1588-1603, 1588-1604, 1589-1604, 1589-1605, 1590-1605, 1590-1606, 1591-1606, 1604-1623, 1607-1626, 1630-1649, 1633-1652, 1645-1664, 1651-1670, 1654-1674, 1657-1676, 1660-1679, 1663-1682, 1666-1685, 1689-1708, 1695-1714, 1698-1717, 1701-1720, 1716-1735, 1778-1797, 1778-1794, 1778-1797, 1779-1795, 1779-1798, 1780-1795, 1780-1796, 1780-1799, 1781-1800, 1794-1813, 1895-1914, 1898-1917, 1901-1920, 1907-1926, 1910-1929, 1913-1932, 1916-1935, 1919-1938, 2278-2297, 2281-2300, and 2284-2303.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 80% inhibition: 13-32, 16-35, 19-38, 25-44, 28-47, 46-65, 49-68, 58-77, 59-80, 63-82, 98-120, 116-135, 137-159, 158-177, 167-186, 203-224, 205-224, 209-228, 218-237, 233-252, 236-263, 245-264, 253-272, 256-275, 257-276, 266-288, 281-300, 290-312, 293-312, 324-343, 339-358, 348-367, 358-378, 360-379, 361-383, 366-385, 373-392, 382-401, 405-424, 411-431, 411-426, 411-427, 411-430, 413-428, 414-433, 414-434, 415-430, 415-434, 416-431, 416-435, 417-436, 418-437, 422-441, 425-444, 434-453, 456-476, 458-473, 458-477, 464-483, 471-493, 488-507, 494-513, 512-531, 524-543, 527-546, 536-558, 560-579, 566-585, 572-591, 575-594, 584-603, 587-606, 608-627, 614-633, 617-636, 620-639, 623-642, 626-645, 629-648, 639-654, 641-656, 642-657, 643-658, 665-688, 670-687, 670-686, 671-686, 671-687, 671-691, 673-688, 679-699, 682-697, 682-706, 686-701, 687-702, 687-706, 687-703, 693-715, 727-746, 742-761, 748-767, 757-776, 766-785, 790-815, 814-833, 820-839, 822-844, 845-864, 854-873, 854-876, 863-885, 872-906, 878-897, 899-918, 905-933, 936-955, 951-979, 963-985, 966-985, 972-1015, 978-997, 1002-1021, 1025-1044, 1031-1056, 1049-1074, 1061-1083, 1070-1089, 1082-1101, 1088-11107, 1094-1119, 1109-1134, 1121-1140, 1127-1146, 1159-1187, 1171-1191, 1206-1228, 1209-1228, 1215-1255, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1279, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1282, 1264-1279, 1264-1283, 1265-1284, 1266-1285, 1267-1282, 1267-1283, 1268-1283, 1268-1284, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1275-1294, 1282-1301, 1286-1306, 1293-1318, 1311-1333, 1326-1345, 1359-1378, 1553-1578, 1565-1584, 1571-1590, 1574-1599, 1577-1592, 1577-1596, 1577-1593, 1577-1596, 1578-1593, 1578-1594, 1578-1597, 1579-1595, 1579-1598, 1580-1596, 1580-1599, 1581-1596, 1581-1597, 1581-1600, 1582-1597, 1582-1601, 1582-1602, 1583-1598, 1583-1599, 1583-1602, 1584-1599, 1584-1603, 1585-1601, 1585-1604, 1586-1605, 1587-1602, 1587-1606, 1588-1603, 1589-1604, 1589-1605, 1657-1679, 1780-1795, 1780-1796, 1780-1799, 1913-1935, 2278-2297, 2281-2300, and 2284-2303.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 85% inhibition: 13-32, 16-35, 19-38, 25-44, 46-65, 59-80, 101-120, 140-159, 158-177, 167-186, 200-219, 205-224, 209-228, 233-252, 242-263, 253-272, 266-285, 281-300, 290-311, 293-312, 359-379, 361-381, 370-389, 382-401, 411-426, 411-430, 411-427, 413-428, 414-433, 415-430, 416-435, 417-436, 422-441, 456-476, 458-473, 470-493, 512-531, 524-543, 536-558, 566-585, 575-594, 587-606, 608-627, 614-636, 623-645, 639-654, 665-687, 671-686, 671-687, 680-699, 682-703, 687-706, 687-703, 727-746, 742-761, 757-776, 793-812, 822-843, 854-876, 854-873, 863-885, 878-900, 878-897, 887-906, 899-918, 905-927, 914-933, 936-955, 951-985, 966-985, 972-1015, 978-997, 1002-1021, 1025-1044, 1037-1056, 1049-1074, 1064-1083, 1070-1089, 1088-1107, 1094-1119, 1109-1128, 1121-1140, 1156-1175, 1162-1187, 1172-1191, 1206-1228, 1209-1228, 1215-1255, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1279, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1276, 1261-1277, 1261-1280, 1262-1277, 1262-1278, 1262-1281, 1263-1278, 1263-1282, 1264-1279, 1264-1283, 1265-1284, 1266-1285, 1267-1282, 1267-1283, 1268-1284, 1269-1284, 1269-1285, 1269-1288, 1270-1285, 1275-1294, 1282-1301, 1293-1315, 1311-1330, 1359-1378, 1574-1593, 1577-1592, 1577-1593, 1577-1596, 1577-1606, 1578-1593, 1578-1594, 1578-1597, 1579-1598, 1580-1596, 1580-1599, 1581-1597, 1581-1600, 1582-1601, 1583-1598, 1583-1602, 1584-1603, 1585-1601, 1585-1604, 1586-1605, 1587-1602, 1588-1603, 1780-1799, 1780-1796, and 2278-2297, 2281-2300, and 2284-2303.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 90% inhibition: 13-32, 16-35, 60-80, 140-159, 158-177, 167-186, 242-261, 292-311, 362-381, 370-389, 382-401, 411-427, 411-426, 413-428, 415-430, 416-435, 422-441, 473-492, 617-636, 623-642, 639-654, 668-687, 680-699, 682-701, 684-703, 687-706, 727-746, 757-776, 824-843, 854-873, 854-876, 863-882, 878-897, 878-900, 887-906, 899-918, 905-927, 914-933, 936-955, 951-970, 960-985, 966-985, 972-1015, 978-997, 1025-1044, 1037-1056, 1070-1089, 1097-1119, 1109-1128, 1121-1140, 1165-1187, 1172-1191, 1206-1228, 1209-1228, 1215-1234, 1215-1234, 1215-1255, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1279, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1280, 1262-1278, 1261-1276, 1262-1281, 1262-1277, 1263-1282, 1263-1278, 1264-1283, 1265-1284, 1266-1285, 1268-1284, 1269-1284, 1269-1285, 1269-1288, 1296-1315, 1577-1605, 1577-1596, 1577-1593, 1577-1592, 1578-1597, 1581-1600, 1582-1601, 1583-1602, 1583-1598, 1585-1601, 1585-1604, 1586-1605, 1588-1603, 1780-1799, 1780-1796, 2278-2297, 2281-2300, and 2284-2303.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 95% inhibition: 411-426, 411-427, 413-428, 617-636, 623-642, 668-687, 680-699, 682-701, 854-873, 878-897, 887-906, 914-933, 966-985, 978-997, 1209-1228, 1215-1234, 1218-1237, 1221-1240, 1224-1243, 1227-1246, 1230-1249, 1233-1252, 1236-1255, 1245-1264, 1251-1270, 1254-1273, 1254-1279, 1257-1276, 1258-1277, 1259-1278, 1260-1279, 1261-1285, 1261-1280, 1262-1281, 1263-1282, 1263-1278, 1264-1283, 1265-1284, 1266-1285, 1268-1284, 1269-1288, 1577-1592, 1577-1596, 1577-1601, 1583-1598, 1585-1601, 1588-1603, 1780-1799, 2278-2297, 2281-2300, and 2284-2303.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 50% inhibition of a HBV mRNA, ISIS IDs: 510088, 510089, 510090, 510092, 510096, 510097, 510098, 510099, 510100, 510101, 510102, 505330, 509928, 510104, 509929, 510105, 509930, 510106, 510107, 510108, 510111, 510115, 509931, 510116, 510117, 510118, 510119, 510120, 510121, 509932, 510122, 509933, 510123, 509934, 510124, 509935, 510125, 510126, 510127, 510128, 510140, 146779, 505314, 505315, 505316, 505317, 146821, 505318, 509922, 505319, 509925, 505320, 509952, 505321, 505322, 505323, 505324, 505325, 505326, 505327, 505328, 505329, 509956, 509957, 509927, 509958, 510038, 505330, 509959, 510039, 509960, 510040, 509961, 510041, 509962, 509963, 505331, 505332, 509968, 509969, 510050, 510052, 505333, 505334, 505335, 505336, 509972, 146823, 509974, 505338, 505339, 509975, 505340, 509978, 505341, 509979, 510058, 505342, 509981, 510061, 505344, 505345, 509983, 505346, 509984, 505347, 505348, 505350, 505352, 505353, 505354, 505355, 505356, 146786, 505357, 505358, 505359, 505360, 509985, 509986, 509987, 509988, 505363, 505364, 505365, 505366, 146787, 510079, 524410, 524411, 524413, 524414, 524415, 524416, 524417, 524418, 524419, 524420, 524421, 524422, 524424, 524425, 524426, 524427, 524428, 524429, 524431, 524432, 524433, 524434, 524435, 524436, 524439, 524440, 524442, 524444, 524446, 524447, 524448, 524450, 524451, 524452, 524453, 524454, 524455, 524456, 524457, 524458, 524459, 524460, 524461, 524462, 524464, 524466, 524467, 524468, 524469, 524470, 524471, 524472, 524473, 524474, 524475, 524477, 524478, 524479, 524480, 524481, 524482, 524483, 524484, 524485, 524486, 524487, 524489, 524490, 524491, 524492, 524493, 524494, 524495, 524496, 524498, 524499, 524500, 524501, 524502, 524503, 524504, 524506, 524507, 524508, 524509, 524510, 524511, 524512, 524513, 524514, 524515, 524516, 524517, 524518, 524519, 524520, 524521, 524522, 524523, 524524, 524525, 524526, 524527, 524528, 524529, 524530, 524531, 524532, 524533, 524534, 524535, 524536, 524537, 524538, 524539, 524540, 524541, 524543, 524544, 524546, 524547, 524548, 524549, 524550, 524551, 524552, 524553, 524554, 524555, 524556, 524557, 524558, 524559, 524560, 524561, 524562, 524563, 524564, 524565, 524568, 524569, 524570, 524571, 524572, 524573, 524574, 524575, 524576, 524577, 524578, 524579, 524580, 524581, 524582, 524584, 524585, 524586, 524587, 524588, 524589, 524590, 524591, 524592, 524593, 524594, 524595, 524598, 524599, 524600, 524601, 524602, 524603, 524604, 524605, 524606, 524607, 524608, 524609, 524610, 524611, 524614, 524615, 524616, 524617, 524618, 524619, 524620, 524621, 524622, 524623, 524624, 524625, 524626, 524627, 524629, 524632, 524633, 524634, 524635, 524636, 524637, 524638, 524639, 524640, 524641, 524642, 524643, 524644, 524646, 524647, 524648, 524649, 524650, 524651, 524652, 524654, 524656, 524657, 524658, 524659, 524660, 524661, 524662, 524663, 524664, 524665, 524666, 524667, 524668, 524669, 524670, 524672, 524673, 524675, 524676, 524678, 524679, 524680, 524682, 524683, 524684, 524685, 524686, 524687, 524688, 524689, 524690, 524691, 524692, 524693, 524694, 524695, 524696, 524697, 524698, 524699, 524700, 524701, 524702, 524703, 524704, 524705, 524706, 524707, 524708, 524709, 524710, 524712, 524713, 524714, 524715, 524716, 524717, 524718, 524719, 524721, 524722, 524723, 524724, 524726, 524727, 524728, 524729, 524730, 524731, 524732, 524733, 524734, 524735, 524736, 524737, 524738, 524739, 524740, 524741, 524742, 524743, 524744, 524745, 524746, 524747, 524748, 524749, 524750, 524751, 524752, 524753, 524754, 524755, 524756, 524757, 524758, 524759, 524760, 524761, 524762, 524763, 524764, 524765, 524766, 524767, 524768, 524769, 524770, 524771, 524772, 524773, 524774, 524775, 524776, 524777, 524778, 524779, 524780, 524781, 524782, 524783, 524784, 524785, 524786, 524787, 524788, 524789, 524790, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524799, 524800, 524801, 524802, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524812, 524813, 524814, 524815, 524816, 524817, 524818, 524819, 524820, 524821, 524822, 524823, 524824, 524825, 524826, 524827, 524828, 524829, 524830, 524831, 524632, 524833, 524834, 524835, 524842, 524843, 524844, 524845, 524847, 524848, 524856, 524857, 524861, 524866, 524867, 524868, 524869, 524870, 524871, 524872, 524873, 524875, 524876, 524877, 524878, 524879, 524880, 524881, 524882, 524883, 524884, 524885, 524886, 524887, 524888, 524889, 524890, 524891, 524892, 524893, 524894, 524895, 524896, 524897, 524898, 524899, 524900, 524901, 524902, 524903, 524904, 524905, 524906, 524907, 524908, 524909, 524910, 524911, 524912, 524913, 524914, 524915, 524916, 524917, 524918, 524919, 524921, 524922, 524923, 524924, 524925, 524926, 524927, 524928, 524929, 524930, 524931, 524932, 524933, 524934, 524935, 524936, 524937, 524938, 524939, 524940, 524941, 524942, 524943, 524944, 524945, 524946, 524947, 524948, 524949, 524950, 524951, 524952, 524953, 524954, 524955, 524956, 524957, 524958, 524959, 524960, 524961, 524962, 524964, 524965, 524976, 524977, 524978, 524979, 524980, 524981, 524982, 524983, 524984, 524985, 524986, 524987, 524988, 524989, 524991, 524992, 524993, 524994, 524997, 524998, 525021, 525022, 525037, 525039, 525043, 525050, 525052, 525086, 525090, 525100, 551909, 551910, 551911, 551912, 551913, 551916, 551917, 551918, 551919, 551920, 551921, 551922, 551923, 551924, 551925, 551926, 551927, 551928, 551929, 551930, 551932, 551933, 551934, 551935, 551936, 551937, 551939, 551940, 551941, 551942, 551943, 551944, 551945, 551946, 551947, 551948, 551949, 551950, 551951, 551952, 551953, 551954, 551955, 551956, 551957, 551958, 551959, 551960, 551962, 551963, 551964, 551965, 551966, 551967, 551968, 551971, 551972, 551973, 551974, 551975, 551976, 551977, 551978, 551979, 551980, 551981, 551982, 551983, 551984, 551985, 551986, 551987, 551988, 551989, 551990, 551992, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552001, 552002, 552003, 552004, 552005, 552006, 552007, 552009, 552010, 552011, 552012, 552013, 552014, 552015, 552016, 552017, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552035, 552036, 552037, 552038, 552039, 552040, 552041, 552042, 552043, 552044, 552045, 552046, 552047, 552048, 552049, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552067, 552068, 552069, 552070, 552071, 552072, 552073, 552074, 552075, 552076, 552077, 552078, 552079, 552080, 552081, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552099, 552100, 552101, 552102, 552114, 552115, 552116, 552117, 552118, 552119, 552122, 552123, 552124, 552125, 552126, 552127, 552128, 552129, 552131, 552132, 552133, 552134, 552135, 552136, 552137, 552138, 552139, 552140, 552141, 552142, 552143, 552144, 552145, 552146, 552147, 552148, 552149, 552150, 552151, 552152, 552153, 552154, 552155, 552158, 552159, 552160, 552161, 552162, 552163, 552164, 552165, 552167, 552168, 552169, 552170, 552171, 552175, 552176, 552177, 552178, 552179, 552180, 552181, 552182, 552183, 552185, 552186, 552187, 552188, 552189, 552191, 552192, 552193, 552194, 552195, 552196, 552197, 552198, 552199, 552200, 552201, 552202, 552203, 552204, 552205, 552206, 552207, 552208, 552209, 552210, 552211, 552212, 552213, 552214, 552215, 552216, 552217, 552218, 552220, 552222, 552224, 552225, 552230, 552239, 552240, 552241, 552242, 552243, 552246, 552247, 552248, 552249, 552250, 552251, 552252, 552253, 552254, 552255, 552256, 552257, 552258, 552259, 552260, 552261, 552262, 552263, 552264, 552265, 552266, 552267, 552268, 552269, 552270, 552271, 552279, 552285, 552288, 552293, 552294, 552295, 552296, 552297, 552300, 552301, 552302, 552303, 552304, 552305, 552306, 552307, 552308, 552309, 552310, 552312, 552313, 552314, 552315, 552316, 552317, 552318, 552319, 552320, 552321, 552322, 552323, 552325, 552326, 552330, 552331, 552332, 552333, 552337, 552338, 552339, 552340, 552341, 552342, 552343, 552344, 552345, 552347, 552348, 552349, 552350, 552351, 552352, 552354, 552355, 552356, 552357, 552358, 552359, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552368, 552369, 552370, 552371, 552372, 552373, 552374, 552375, 552376, 552377, 552378, 552379, 552380, 552385, 552386, 552390, 552391, 552393, 552394, 552395, 552396, 552397, 552398, 552399, 552400, 552401, 552402, 552403, 552408, 552409, 552410, 552411, 552412, 552413, 552414, 552415, 552416, 552417, 552418, 552419, 552420, 552421, 552422, 552423, 552424, 552425, 552428, 552430, 552431, 552432, 552433, 552440, 552442, 552443, 552444, 552445, 552446, 552447, 552448, 552449, 552450, 552452, 552453, 552455, 552456, 552458, 552459, 552464, 552465, 552466, 552467, 552468, 552469, 552470, 552471, 552472, 552473, 552474, 552475, 552476, 552477, 552478, 552479, 552480, 552481, 552482, 552484, 552485, 552486, 552487, 552488, 552490, 552491, 552493, 552497, 552499, 552500, 552501, 552502, 552503, 552504, 552505, 552506, 552508, 552509, 552510, 552511, 552512, 552513, 552514, 552515, 552516, 552517, 552520, 552521, 552522, 552523, 552525, 552526, 552527, 552528, 552529, 552530, 552531, 552532, 552533, 552534, 552535, 552538, 552539, 552540, 552541, 552542, 552544, 552547, 552548, 552553, 552554, 552555, 552557, 552558, 552559, 552561, 552562, 552565, 552566, 552567, 552568, 552569, 552570, 552571, 552572, 552576, 552577, 552578, 552579, 552580, 552581, 552582, 552583, 552584, 552585, 552586, 552587, 552588, 552589, 552590, 552591, 552592, 552594, 552595, 552596, 552597, 552598, 552600, 552606, 552608, 552787, 552788, 552789, 552790, 552791, 552794, 552795, 552796, 552797, 552798, 552799, 552800, 552801, 552802, 552803, 552804, 552805, 552806, 552807, 552808, 552809, 552810, 552811, 552812, 552813, 552814, 552815, 552816, 552817, 552818, 552819, 552820, 552821, 552822, 552823, 552824, 552825, 552826, 552827, 552828, 552829, 552830, 552831, 552832, 552833, 552834, 552835, 552836, 552837, 552838, 552839, 552840, 552841, 552842, 552843, 552844, 552845, 552846, 552847, 552848, 552849, 552850, 552851, 552852, 552853, 552854, 552855, 552856, 552857, 552858, 552859, 552860, 552861, 552862, 552863, 552864, 552865, 552866, 552868, 552870, 552871, 552872, 552876, 552889, 552890, 552891, 552892, 552893, 552894, 552895, 552896, 552898, 552899, 552901, 552902, 552903, 552904, 552905, 552907, 552908, 552909, 552910, 552911, 552912, 552913, 552914, 552915, 552916, 552917, 552918, 552919, 552922, 552923, 552925, 552926, 552927, 552928, 552929, 552930, 552931, 552932, 552933, 552934, 552935, 552936, 552937, 552938, 552939, 552940, 552941, 552942, 552943, 552944, 552945, 552946, 552947, 552948, 552950, 552951, 552953, 552954, 552955, 552956, 552957, 552958, 552959, 552960, 552961, 552965, 552966, 552969, 552970, 552971, 552972, 552973, 552974, 552975, 552976, 552977, 552979, 552980, 552981, 552982, 552983, 552984, 552987, 552988, 552989, 552990, 552991, 552992, 552993, 552994, 552995, 552996, 552997, 552998, 552999, 553000, 553001, 553002, 553003, 553004, 553005, 553006, 553007, 553008, 553009, 553010, 553011, 553012, 553014, 553015, 553016, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577129, 577130, 577131, 577132, 577133, 577134, 577135, 577136, 582665, and 582666.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 50% inhibition of a HBV mRNA, SEQ ID NOs: 5, 6, 7, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 33, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 74, 83, 85, 86, 87, 88, 89, 92, 96, 98, 99, 100, 102, 103, 104, 106, 108, 109, 111, 112, 115, 117, 121, 122, 123, 124, 125, 126, 127, 128, 136, 137, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 155, 157, 159, 161, 165, 166, 167, 168, 169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 186, 187, 188, 189, 190, 191, 192, 193, 194, 197, 198, 199, 201, 203, 206, 207, 208, 209, 210, 211, 212, 213, 215, 217, 218, 220, 221, 222, 224, 225, 226, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 240, 241, 242, 243, 244, 250, 283, 321, 322, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335, 336, 337, 338, 339, 340, 342, 343, 344, 345, 346, 347, 350, 351, 353, 355, 357, 358, 359, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 399, 400, 401, 402, 403, 404, 405, 406, 408, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 420 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 473, 474, 475, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 539, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 564, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 582, 583, 585, 586, 588, 589, 590, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 622, 623, 624, 625, 626, 627, 628, 629, 631, 632, 633, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 754, 755, 756, 757, 759, 760, 768, 769, 773, 777, 778, 779, 780, 781, 782, 783, 784, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 876, 877, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 903, 904, 905, 906, 909, 910, 933, 934, 949, 951, 955, 962, 964, 998, 1002, 1013, 1052, 1267, 1271, 1272, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1375, and 1376.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 60% inhibition of a HBV mRNA, ISIS IDs: 510090, 510100, 510102, 505330, 509928, 510104, 509929, 510105, 509930, 510106, 510107, 510111, 509931, 510116, 510117, 510118, 510119, 510120, 510121, 509932, 510122, 509933, 510123, 509934, 510124, 509935, 510125, 510128, 146779, 505314, 505315, 505316, 505317, 146821, 505318, 505319, 505322, 505323, 505324, 505325, 505326, 505327, 505328, 505329, 509956, 509957, 509958, 505330, 509959, 510041, 505332, 509968, 505333, 505335, 146823, 509974, 505338, 505339, 509975, 505340, 505341, 509979, 505342, 509981, 505344, 505345, 509983, 505346, 509984, 505347, 505348, 505353, 505354, 505356, 146786, 505357, 505358, 505359, 505360, 509985, 509986, 505363, 505366, 524410, 524413, 524414, 524415, 524416, 524417, 524418, 524419, 524420, 524421, 524422, 524424, 524425, 524426, 524428, 524431, 524432, 524433, 524434, 524435, 524439, 524440, 524446, 524447, 524448, 524451, 524452, 524453, 524454, 524455, 524456, 524457, 524459, 524460, 524461, 524464, 524466, 524467, 524468, 524469, 524471, 524472, 524473, 524474, 524475, 524477, 524478, 524479, 524480, 524481, 524482, 524485, 524486, 524487, 524489, 524490, 524491, 524492, 524493, 524494, 524495, 524496, 524499, 524500, 524501, 524502, 524503, 524504, 524506, 524507, 524508, 524509, 524510, 524511, 524512, 524513, 524514, 524515, 524516, 524517, 524519, 524520, 524521, 524523, 524525, 524526, 524527, 524528, 524529, 524532, 524533, 524534, 524535, 524536, 524537, 524538, 524539, 524540, 524541, 524543, 524546, 524547, 524549, 524550, 524552, 524553, 524554, 524555, 524556, 524557, 524558, 524559, 524560, 524561, 524562, 524563, 524564, 524565, 524568, 524569, 524570, 524571, 524572, 524573, 524574, 524575, 524576, 524577, 524578, 524579, 524580, 524581, 524582, 524585, 524586, 524587, 524588, 524589, 524590, 524591, 524593, 524594, 524595, 524598, 524599, 524600, 524602, 524603, 524604, 524605, 524606, 524607, 524610, 524611, 524614, 524615, 524616, 524617, 524618, 524619, 524620, 524621, 524622, 524623, 524625, 524627, 524629, 524632, 524633, 524634, 524635, 524636, 524637, 524638, 524639, 524640, 524641, 524642, 524643, 524644, 524646, 524647, 524648, 524649, 524650, 524651, 524654, 524656, 524657, 524658, 524659, 524661, 524662, 524663, 524664, 524665, 524666, 524667, 524668, 524669, 524670, 524673, 524675, 524676, 524678, 524679, 524680, 524683, 524684, 524685, 524686, 524687, 524688, 524689, 524690, 524691, 524692, 524694, 524695, 524696, 524697, 524698, 524699, 524700, 524701, 524702, 524703, 524704, 524705, 524706, 524707, 524708, 524709, 524710, 524713, 524714, 524715, 524716, 524717, 524718, 524719, 524721, 524722, 524724, 524726, 524727, 524728, 524729, 524730, 524731, 524732, 524733, 524734, 524735, 524736, 524737, 524738, 524739, 524741, 524742, 524743, 524744, 524746, 524747, 524748, 524749, 524750, 524751, 524752, 524753, 524754, 524755, 524756, 524757, 524758, 524759, 524760, 524761, 524762, 524763, 524764, 524765, 524766, 524767, 524768, 524769, 524770, 524771, 524772, 524773, 524774, 524775, 524776, 524777, 524778, 524779, 524780, 524781, 524782, 524783, 524784, 524785, 524787, 524788, 524789, 524790, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524799, 524800, 524801, 524802, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524812, 524813, 524814, 524815, 524816, 524817, 524818, 524819, 524820, 524821, 524822, 524823, 524824, 524825, 524826, 524827, 524828, 524829, 524830, 524632, 524833, 524842, 524843, 524844, 524845, 524847, 524856, 524866, 524867, 524868, 524869, 524870, 524871, 524872, 524873, 524876, 524878, 524879, 524880, 524881, 524882, 524883, 524884, 524885, 524886, 524887, 524888, 524889, 524890, 524891, 524892, 524893, 524894, 524895, 524896, 524897, 524898, 524899, 524900, 524901, 524902, 524903, 524904, 524905, 524906, 524907, 524908, 524909, 524910, 524911, 524912, 524913, 524914, 524915, 524916, 524921, 524922, 524923, 524924, 524925, 524926, 524928, 524929, 524930, 524931, 524932, 524933, 524936, 524937, 524938, 524939, 524940, 524941, 524942, 524944, 524946, 524947, 524948, 524949, 524950, 524952, 524953, 524954, 524955, 524961, 524977, 524978, 524979, 524980, 524981, 524982, 524983, 524984, 524985, 524986, 524987, 524988, 524991, 524992, 524993, 524994, 525037, 525052, 551909, 551911, 551919, 551920, 551921, 551922, 551924, 551925, 551926, 551927, 551928, 551932, 551933, 551934, 551935, 551936, 551941, 551943, 551944, 551948, 551949, 551950, 551951, 551952, 551953, 551954, 551955, 551956, 551957, 551958, 551959, 551960, 551962, 551963, 551965, 551966, 551967, 551968, 551973, 551975, 551979, 551981, 551982, 551983, 551984, 551985, 551986, 551987, 551989, 551990, 551992, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552001, 552002, 552003, 552005, 552006, 552007, 552009, 552010, 552012, 552013, 552014, 552015, 552016, 552017, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552035, 552036, 552038, 552039, 552041, 552042, 552044, 552045, 552046, 552047, 552048, 552049, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552068, 552069, 552070, 552071, 552073, 552074, 552075, 552076, 552077, 552078, 552079, 552080, 552081, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552099, 552100, 552101, 552102, 552114, 552115, 552116, 552117, 552118, 552119, 552123, 552124, 552125, 552126, 552127, 552128, 552129, 552131, 552132, 552133, 552134, 552135, 552136, 552138, 552139, 552140, 552141, 552143, 552144, 552145, 552146, 552147, 552148, 552149, 552150, 552151, 552152, 552153, 552155, 552158, 552159, 552160, 552162, 552163, 552168, 552169, 552170, 552171, 552176, 552178, 552179, 552180, 552182, 552183, 552185, 552187, 552188, 552191, 552192, 552193, 552194, 552195, 552196, 552197, 552198, 552199, 552200, 552201, 552202, 552203, 552204, 552205, 552206, 552207, 552208, 552209, 552210, 552211, 552212, 552213, 552214, 552215, 552216, 552222, 552224, 552225, 552239, 552240, 552242, 552246, 552247, 552248, 552252, 552253, 552254, 552255, 552256, 552257, 552258, 552259, 552261, 552263, 552265, 552266, 552268, 552285, 552293, 552294, 552295, 552296, 552301, 552302, 552303, 552306, 552307, 552308, 552309, 552310, 552312, 552313, 552314, 552315, 552316, 552317, 552318, 552320, 552321, 552322, 552323, 552325, 552326, 552331, 552332, 552337, 552338, 552339, 552340, 552343, 552345, 552347, 552348, 552349, 552351, 552354, 552355, 552356, 552358, 552359, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552368, 552369, 552370, 552371, 552372, 552373, 552374, 552375, 552376, 552377, 552378, 552379, 552396, 552397, 552398, 552403, 552408, 552409, 552410, 552411, 552412, 552414, 552416, 552418, 552419, 552420, 552421, 552422, 552423, 552424, 552431, 552442, 552445, 552449, 552455, 552456, 552459, 552464, 552465, 552466, 552467, 552469, 552472, 552473, 552474, 552475, 552477, 552478, 552479, 552480, 552484, 552487, 552497, 552508, 552509, 552511, 552512, 552515, 552516, 552520, 552521, 552522, 552523, 552526, 552527, 552528, 552529, 552530, 552531, 552534, 552540, 552541, 552542, 552559, 552567, 552568, 552569, 552570, 552572, 552576, 552577, 552578, 552579, 552582, 552583, 552584, 552585, 552586, 552587, 552588, 552590, 552595, 552596, and 552597, 552788, 552789, 552790, 552791, 552796, 552800, 552801, 552803, 552804, 552805, 552806, 552807, 552808, 552809, 552811, 552812, 552813, 552814, 552815, 552816, 552817, 552818, 552819, 552820, 552821, 552822, 552823, 552824, 552826, 552827, 552828, 552829, 552830, 552831, 552832, 552833, 552834, 552835, 552836, 552837, 552838, 552839, 552841, 552842, 552843, 552844, 552845, 552846, 552847, 552848, 552849, 552850, 552851, 552852, 552853, 552854, 552855, 552856, 552857, 552858, 552859, 552860, 552861, 552862, 552863, 552864, 552865, 552866, 552872, 552891, 552892, 552893, 552894, 552902, 552903, 552904, 552905, 552907, 552908, 552909, 552910, 552911, 552912, 552913, 552914, 552915, 552916, 552917, 552918, 552922, 552923, 552925, 552927, 552928, 552929, 552930, 552931, 552932, 552933, 552934, 552935, 552936, 552937, 552938, 552939, 552940, 552941, 552942, 552943, 552944, 552945, 552946, 552951, 552955, 552956, 552957, 552958, 552960, 552961, 552966, 552969, 552971, 552972, 552973, 552974, 552975, 552976, 552977, 552979, 552980, 552981, 552982, 552983, 552984, 552988, 552989, 552990, 552991, 552992, 552993, 552994, 552995, 552996, 552998, 552999, 553000, 553001, 553002, 553003, 553004, 553005, 553006, 553007, 553008, 553009, 553010, 553011, 553012, 553016, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577129, 577130, 577131, 577132, 577133, 577134, 577135, 577136, and 582666.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 60% inhibition of a HBV mRNA, SEQ ID NOs: 7, 9, 10, 12, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 33, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 83, 85, 86, 87, 88, 89, 92, 96, 98, 100, 102, 103, 112, 115, 117, 122, 123, 124, 125, 126, 127, 128, 136, 137, 139, 140, 142, 143, 145, 147, 149, 150, 151, 153, 155, 157, 159, 161, 166, 167, 168, 172, 174, 176, 177, 178, 179, 180, 181, 186, 187, 188, 189, 190, 191, 192, 193, 194, 198, 199, 201, 206, 207, 208, 209, 210, 211, 212, 213, 218, 220, 222, 224, 225, 226, 227, 228, 230, 231, 232, 233, 234, 240, 243, 321, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 335, 336, 337, 339, 342, 343, 344, 345, 346, 350, 351, 357, 358, 359, 362, 363, 364, 365, 366, 367, 368, 370, 371, 372, 375, 376, 377, 378, 379, 381, 382, 383, 384, 387, 388, 389, 390, 391, 392, 395, 396, 397, 399, 400, 401, 402, 403, 404, 405, 406, 409, 410, 411, 412, 413, 414, 416, 417, 418, 419, 420 421, 422, 423, 424, 425, 426, 427, 429, 430, 431, 433, 435, 436, 437, 438, 439, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 456, 457, 459, 460, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 473, 474, 475, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 508, 509, 510, 512, 513, 514, 515, 516, 517, 520, 521, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 535, 537, 539, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 564, 566, 567, 568, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 583, 585, 586, 588, 589, 590, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 623, 624, 625, 626, 627, 628, 629, 631, 632, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 740, 741, 742, 744, 745, 754, 755, 756, 757, 759, 768, 777, 778, 779, 780, 781, 782, 783, 784, 787, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 833, 834, 835, 836, 837, 838, 840, 841, 842, 843, 844, 845, 848, 849, 850, 851, 852, 853, 854, 856, 858, 859, 860, 861, 862, 864, 865, 866, 867, 873, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 903, 904, 905, 906, 949, 964, 1271, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, and 1376.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 70% inhibition of a HBV mRNA, ISIS IDs: 510100, 505330, 509928, 509929, 509930, 510106, 509931, 510116, 510119, 510120, 510121, 509932, 510122, 509933, 510123, 509934, 510124, 509935, 146779, 505317, 146821, 505318, 505319, 505323, 505325, 505326, 505327, 509957, 505330, 505332, 505335, 509974, 505338, 505339, 509975, 505342, 509981, 505345, 505346, 505347, 505348, 146786, 505357, 505358, 505359, 505363, 524410, 524413, 524414, 524415, 524416, 524418, 524419, 524420, 524421, 524424, 524425, 524426, 524428, 524431, 524432, 524433, 524434, 524435, 524446, 524447, 524448, 524452, 524453, 524457, 524459, 524460, 524461, 524464, 524466, 524467, 524468, 524469, 524472, 524473, 524474, 524475, 524477, 524478, 524479, 524480, 524481, 524482, 524485, 524487, 524490, 524491, 524492, 524493, 524494, 524495, 524499, 524500, 524502, 524503, 524507, 524508, 524510, 524511, 524512, 524513, 524514, 524515, 524516, 524517, 524520, 524525, 524526, 524528, 524532, 524533, 524534, 524535, 524536, 524537, 524538, 524539, 524540, 524541, 524547, 524549, 524552, 524553, 524554, 524555, 524556, 524557, 524558, 524559, 524560, 524561, 524563, 524564, 524565, 524568, 524569, 524570, 524571, 524572, 524573, 524574, 524575, 524577, 524578, 524579, 524580, 524582, 524586, 524587, 524590, 524591, 524594, 524595, 524598, 524600, 524602, 524603, 524604, 524605, 524606, 524607, 524610, 524611, 524614, 524615, 524616, 524617, 524618, 524619, 524620, 524621, 524629, 524633, 524634, 524635, 524636, 524637, 524638, 524641, 524642, 524643, 524644, 524646, 524647, 524648, 524649, 524650, 524651, 524656, 524657, 524659, 524661, 524662, 524663, 524664, 524665, 524666, 524667, 524668, 524669, 524670, 524678, 524679, 524680, 524685, 524686, 524687, 524688, 524689, 524690, 524691, 524692, 524695, 524696, 524698, 524699, 524700, 524701, 524702, 524703, 524704, 524705, 524706, 524707, 524708, 524709, 524713, 524714, 524715, 524716, 524717, 524718, 524721, 524722, 524724, 524726, 524727, 524728, 524729, 524730, 524731, 524732, 524733, 524734, 524735, 524736, 524737, 524738, 524739, 524741, 524742, 524743, 524746, 524747, 524748, 524749, 524750, 524751, 524752, 524754, 524755, 524756, 524758, 524760, 524761, 524762, 524763, 524764, 524765, 524766, 524767, 524768, 524769, 524771, 524773, 524775, 524776, 524777, 524778, 524779, 524780, 524781, 524782, 524783, 524784, 524785, 524787, 524788, 524789, 524790, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524799, 524800, 524801, 524802, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524812, 524813, 524814, 524815, 524816, 524817, 524818, 524819, 524821, 524822, 524823, 524824, 524825, 524826, 524827, 524828, 524829, 524830, 524833, 524842, 524843, 524844, 524845, 524856, 524866, 524867, 524868, 524869, 524870, 524871, 524873, 524879, 524880, 524881, 524882, 524883, 524884, 524885, 524886, 524887, 524888, 524889, 524890, 524891, 524892, 524893, 524894, 524895, 524896, 524897, 524898, 524899, 524900, 524902, 524903, 524905, 524906, 524907, 524908, 524909, 524910, 524911, 524912, 524913, 524914, 524915, 524916, 524921, 524922, 524930, 524931, 524932, 524937, 524940, 524942, 524948, 524980, 524981, 524982, 524983, 524984, 524985, 524986, 524987, 524988, 551919, 551921, 551922, 551924, 551925, 551926, 551933, 551941, 551950, 551951, 551952, 551953, 551955, 551956, 551957, 551958, 551966, 551983, 551984, 551985, 551986, 551987, 551989, 551990, 551992, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552005, 552006, 552009, 552012, 552013, 552014, 552015, 552017, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552038, 552039, 552041, 552044, 552046, 552047, 552049, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552068, 552069, 552070, 552071, 552073, 552074, 552075, 552076, 552077, 552078, 552079, 552080, 552081, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552099, 552100, 552101, 552115, 552117, 552123, 552125, 552127, 552128, 552129, 552132, 552133, 552138, 552139, 552140, 552141, 552143, 552144, 552145, 552146, 552147, 552148, 552149, 552150, 552151, 552152, 552158, 552159, 552160, 552163, 552168, 552179, 552187, 552188, 552192, 552193, 552195, 552199, 552200, 552201, 552202, 552203, 552204, 552205, 552206, 552207, 552208, 552210, 552211, 552213, 552214, 552222, 552246, 552247, 552248, 552253, 552254, 552255, 552258, 552294, 552301, 552302, 552306, 552307, 552308, 552309, 552310, 552312, 552314, 552315, 552317, 552318, 552321, 552322, 552323, 552325, 552332, 552337, 552339, 552347, 552348, 552349, 552354, 552355, 552358, 552359, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552368, 552369, 552371, 552373, 552374, 552375, 552376, 552377, 552378, 552379, 552403, 552408, 552409, 552411, 552418, 552419, 552420, 552424, 552442, 552464, 552465, 552466, 552467, 552472, 552474, 552475, 552477, 552478, 552521, 552522, 552523, 552527, 552528, 552529, 552530, 552534, 552567, 552578, 552579, 552584, 552586, 552587, 552588, 552590, 552789, 552803, 552804, 552805, 552808, 552816, 552817, 552818, 552819, 552820, 552821, 552822, 552823, 552824, 552828, 552829, 552830, 552833, 552834, 552835, 552842, 552843, 552844, 552846, 552848, 552849, 552850, 552851, 552852, 552853, 552854, 552855, 552856, 552857, 552858, 552859, 552860, 552861, 552863, 552864, 552865, 552872, 552894, 552903, 552904, 552907, 552909, 552910, 552911, 552913, 552914, 552915, 552916, 552917, 552918, 552922, 552923, 552925, 552927, 552928, 552929, 552930, 552931, 552932, 552933, 552934, 552935, 552936, 552937, 552938, 552939, 552940, 552941, 552942, 552943, 552944, 552945, 552946, 552957, 552961, 552966, 552969, 552971, 552972, 552974, 552976, 552979, 552980, 552981, 552983, 552984, 552988, 552989, 552990, 552991, 552995, 552996, 552998, 552999, 553001, 553002, 553003, 553004, 553006, 553008, 553009, 553010, 553011, 553012, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577129, 577130, 577131, 577132, 577133, 577134, 577135, 577136, and 582666.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 70% inhibition of a HBV mRNA, SEQ ID NOs: 12, 17, 18, 20, 21, 22, 24, 25, 26, 27, 28, 29, 39, 40, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 83, 89, 92, 96, 98, 100, 103, 112, 123, 125, 126, 127, 136, 137, 139, 140, 142, 143, 145, 147, 149, 151, 153, 166, 167, 168, 174, 176, 177, 178, 179, 181, 186, 187, 188, 190, 198, 201, 207, 209, 210, 211, 212, 213, 224, 225, 226, 227, 232, 234, 240, 321, 324, 325, 326, 327, 329, 330, 331, 332, 335, 336, 337, 339, 342, 343, 344, 345, 346, 357, 358, 359, 363, 364, 368, 370, 371, 372, 375, 376, 377, 378, 379, 382, 383, 384, 387, 388, 389, 390, 391, 392, 395, 397, 400, 401, 402, 403, 404, 405, 409, 410, 412, 413, 417, 418, 420 421, 422, 423, 424, 425, 426, 427, 430, 435, 436, 438, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 457, 459, 462, 463, 464, 465, 466, 467, 468, 469, 470, 473, 474, 475, 478, 479, 480, 481, 482, 483, 484, 485, 487, 488, 489, 490, 492, 496, 497, 500, 501, 504, 505, 508, 510, 512, 513, 514, 515, 516, 517, 520, 521, 524, 525, 526, 527, 528, 529, 530, 531, 539, 543, 544, 545, 546, 547, 548, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 566, 567, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 588, 589, 590, 595, 596, 597, 598, 599, 600, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 623, 624, 625, 626, 627, 628, 631, 632, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 657, 658, 659, 660, 661, 662, 663, 665, 666, 667, 669, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 682, 684, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 733, 734, 735, 736, 737, 738, 740, 741, 742, 745, 754, 755, 756, 757, 768, 777, 778, 779, 780, 781, 782, 784, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 814, 815, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 833, 834, 842, 843, 844, 849, 852, 854, 860, 892, 893, 894, 895, 896, 897, 898, 899, 900, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1320, 1322, 1323, 1324, 1325, 1326, 1327, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, and 1350, 1367, 1368, 1369, 1370, 1372, and 1376.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 80% inhibition of a HBV mRNA, ISIS IDs: 510100, 509931, 510116, 505317, 505319, 505323, 505326, 505327, 505330, 505339, 505346, 505347, 505358, 509934, 146786, 524414, 524415, 524416, 524418, 524419, 524425, 524426, 524431, 524432, 524434, 524446, 524447, 524452, 524459, 524460, 524466, 524469, 524475, 524477, 524478, 524479, 524482, 524485, 524490, 524491, 524492, 524493, 524494, 524495, 524499, 524502, 524503, 524507, 524510, 524511, 524512, 524520, 524525, 524528, 524532, 524533, 524534, 524535, 524536, 524540, 524541, 524547, 524552, 524553, 524556, 524561, 524564, 524565, 524568, 524570, 524571, 524572, 524573, 524578, 524580, 524586, 524590, 524591, 524594, 524595, 524602, 524604, 524606, 524607, 524610, 524611, 524614, 524616, 524617, 524618, 524619, 524620, 524621, 524633, 524634, 524635, 524636, 524637, 524641, 524643, 524644, 524646, 524649, 524650, 524651, 524657, 524662, 524664, 524667, 524670, 524678, 524679, 524680, 524686, 524688, 524690, 524691, 524692, 524695, 524698, 524699, 524701, 524702, 524704, 524705, 524706, 524707, 524708, 524709, 524713, 524715, 524716, 524717, 524718, 524721, 524726, 524727, 524728, 524729, 524730, 524731, 524733, 524734, 524735, 524737, 524739, 524741, 524742, 524743, 524747, 524748, 524749, 524751, 524752, 524754, 524758, 524760, 524762, 524763, 524764, 524767, 524768, 524769, 524771, 524773, 524777, 524778, 524779, 524780, 524781, 524783, 524784, 524788, 524789, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524801, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524813, 524816, 524819, 524822, 524823, 524824, 524827, 524828, 524829, 524833, 524842, 524844, 524880, 524881, 524882, 524884, 524886, 524887, 524888, 524889, 524890, 524891, 524893, 524907, 524908, 524980, 524986, 524987, 551921, 551924, 551925, 551953, 551956, 551957, 551984, 551986, 551987, 551989, 551990, 551993, 551994, 551995, 551996, 551997, 551998, 551999, 552000, 552005, 552006, 552018, 552019, 552020, 552021, 552022, 552023, 552024, 552025, 552026, 552027, 552028, 552029, 552030, 552031, 552032, 552033, 552034, 552039, 552044, 552046, 552050, 552051, 552052, 552053, 552054, 552055, 552056, 552057, 552058, 552059, 552060, 552061, 552062, 552063, 552064, 552065, 552073, 552077, 552078, 552079, 552080, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552090, 552091, 552092, 552093, 552094, 552095, 552096, 552097, 552098, 552138, 552139, 552145, 552146, 552147, 552149, 552192, 552193, 552199, 552200, 552201, 552207, 552246, 552247, 552253, 552301, 552307, 552308, 552310, 552317, 552347, 552348, 552354, 552355, 552360, 552361, 552362, 552363, 552364, 552365, 552366, 552367, 552371, 552375, 552464, 552465, 552521, 552808, 552816, 552817, 552818, 552819, 552820, 552822, 552824, 552834, 552844, 552849, 552850, 552851, 552852, 552853, 552854, 552916, 552922, 552923, 552925, 552930, 552931, 552932, 552933, 552936, 552937, 552938, 552939, 552942, 552943, 552944, 552980, 552988, 552989, 552996, 552998, 553002, 553003, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577130, 577131, 577132, 577133, 577134, 577135, 577136, and 582666.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 80% inhibition of a HBV mRNA, SEQ ID NOs: 17, 20, 22, 24, 26, 28, 39, 40, 50, 51, 83, 89, 103, 123, 126, 127, 136, 137, 143, 147, 149, 168, 176, 177, 178, 179, 187, 188, 210, 211, 212, 224, 225, 226, 227, 232, 325, 326, 327, 329, 330, 336, 337, 342, 343, 345, 357, 358, 363, 370, 371, 376, 379, 387, 388, 389, 392, 395, 400, 401, 402, 403, 404, 405, 409, 412, 413, 417, 420 421, 422, 430, 435, 438, 442, 443, 444, 445, 446, 450, 451, 457, 462, 463, 466, 474, 475, 478, 480, 481, 482, 483, 488, 490, 496, 500, 501, 504, 505, 512, 514, 516, 517, 520, 521, 524, 526, 527, 528, 529, 530, 531, 543, 544, 545, 546, 547, 551, 553, 554, 555, 559, 560, 561, 567, 572, 574, 577, 580, 588, 589, 590, 596, 598, 600, 601, 602, 605, 608, 609, 611, 612, 614, 615, 616, 617, 618, 619, 623, 625, 626, 627, 628, 631, 636, 637, 638, 639, 640, 641, 643, 644, 645, 646, 648, 650, 652, 653, 654, 658, 659, 660, 662, 663, 665, 669, 671, 673, 674, 675, 678, 679, 680, 682, 684, 688, 689, 690, 691, 692, 694, 695, 699, 700, 702, 703, 704, 705, 706, 707, 708, 709, 712, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 725, 728, 731, 734, 735, 736, 740, 741, 745, 756, 791, 792, 793, 795, 797, 798, 799, 800, 801, 802, 804, 805, 806, 807, 819, 820, 892, 898, 899, 1292, 1293, 1295, 1296, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1310, 1312, 1316, 1322, 1324, 1325, 1326, 1327, 1330, 1331, 1332, 1333, 1334, 1335, 1338, 1339, 1340, 1341, 1344, 1345, 1349, 1350, 1368, 1372, and 1376.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 90% inhibition of a HBV mRNA, ISIS IDs: 524414, 524415, 524432, 524460, 524466, 524469, 524475, 524477, 524493, 524512, 524535, 524540, 524552, 524561, 524572, 524617, 524619, 524634, 524641, 524644, 524657, 524667, 524691, 524698, 524699, 524701, 524706, 524707, 524709, 524713, 524715, 524716, 524718, 524721, 524726, 524729, 524730, 524731, 524733, 524734, 524735, 524739, 524743, 524754, 524763, 524764, 524767, 524771, 524780, 524781, 524784, 524788, 524789, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524801, 524803, 524804, 524805, 524806, 524807, 524808, 524809, 524810, 524811, 524822, 524827, 524842, 551986, 551987, 551989, 552005, 552018, 552019, 552020, 552021, 552022, 552023, 552025, 552046, 552050, 552051, 552052, 552053, 552054, 552055, 552057, 552082, 552083, 552084, 552085, 552086, 552087, 552088, 552089, 552092, 552093, 552096, 552097, 552307, 552317, 552355, 552361, 552362, 552363, 552817, 552851, 552922, 552923, 566828, 566829, 566830, 566831, 566832, 577120, 577121, 577122, 577123, 577124, 577125, 577126, 577127, 577128, 577130, 577131, 577132, 577134, 577135, 577136, and 582666.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 90% inhibition of a HBV mRNA, SEQ ID NOs: 17, 24, 50, 51, 137, 143, 147, 176, 211, 212, 224, 226, 227, 325, 326, 343, 371, 376, 379, 403, 422, 445, 450, 462, 482, 527, 529, 544, 551, 554, 567, 577, 601, 608, 609, 611, 616, 617, 619, 623, 625, 626, 628, 631, 636, 639, 640, 641, 643, 644, 645, 646, 650, 654, 665, 674, 675, 678, 682, 691, 692, 695, 699, 700, 702, 703, 704, 705, 706, 707, 708, 709, 712, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 735, 801, 804, 805, 807, 1296, 1302, 1303, 1304, 1312, 1325, 1326, 1332, 1334, 1340, 1345, 1349, and 1376.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 95% inhibition of a HBV mRNA, ISIS IDs: 524619, 524634, 524641, 505339, 524698, 524709, 524718, 524731, 524734, 524789, 524791, 524792, 524793, 524794, 524795, 524796, 524797, 524798, 524801, 524803, 524804, 524805, 524806, 505346, 146785, 524807, 505347, 524808, 524809, 524810, 524811, 146786, 525101, 525102, 525103, 525107, 525108, 525109, 525110, 525111, 525112, 525113, 525114, 525115, 525116, 525117, 525118, 525119, 525120, 552018, 552050, 552019, 552051, 552020, 552052, 551987, 552021, 552053, 552005, 552022, 552054, 551989, 552023, 552055, 552084, 552085, 552086, 552087, 552088, 552361, 552317, 566831, 577123, 577124, 566830, 566828, 566829, 577127, 577135, 577132, 577136, 566832, and 577122.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a HBV nucleic acid and effect at least a 95% inhibition of a HBV mRNA, SEQ ID NOs: 17, 50, 137, 143, 187, 210, 212, 224, 529, 544, 551, 608, 619, 628, 641, 645, 700, 702, 703, 704, 705, 706, 707, 708, 709, 712, 715, 716, 717, 718, 719, 720, 721, 722, 723, 1014, 1015, 1016, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1236, 1302, 1312, 1334, 1340, 1345, 1349.

Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, and 1379.

Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, 1376, and 1379, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar and/or constrained ethyl (cEt) sugar. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, or 4 sugar modified nucleosides.

Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, 1288-1350, 1364-1372, 1375, 1376, and 1379.

Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, and 1376, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar and/or constrained ethyl (cEt) sugar. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides.

Certain embodiments provide a method of preventing, ameliorating or treating an HBV-related disease, disorder or condition in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, and 1376, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar and/or constrained ethyl (cEt) sugar. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides.

Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272.

Certain embodiments provide methods of treating HBV related disease, disorder, or condition in an animal, comprising administering to an animal in need thereof a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar.

In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides.

Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, or 1288-1350.

Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272.

Certain embodiments provide a method of reducing HBV expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar.

In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides.

Certain embodiments provide a method of preventing, ameliorating or treating an HBV-related disease, disorder or condition in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, 804-1272, or 1288-1350, wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5-310, 321-802, or 804-1272. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar. In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, or 5 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 9 or 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, or 6 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 2, 3, 4, 5, 6, 7, or 8 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides.

Examples of HBV-related diseases, disorders or conditions include, but are not limited to chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, and conditions having symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen.

Certain embodiments provide a method of reducing HBV mRNA expression in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, reduction of HBV mRNA expression in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, reduction of HBV mRNA expression in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV mRNA expression is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

Certain embodiments provide a method of reducing HBV protein levels in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, reduction of HBV protein levels in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, reduction of HBV protein levels in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV protein level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

Certain embodiments provide a method of reducing HBV DNA levels in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, reduction of HBV DNA levels in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, the mammal may be human, and the hepatitis B virus may be a human hepatitis B virus. More particularly, the human hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America). In certain embodiments, reduction of HBV DNA levels in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV DNA level is reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

Certain embodiments provide a method of reducing HBV antigen levels in an animal comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, reduction of HBV antigen levels in an animal prevents, ameliorates or treats an HBV-related disease, disorder or condition. In certain embodiments, reduction of HBV antigen levels in an animal prevents, ameliorates or treats liver disease. In certain embodiments, the HBV antigen levels are reduced by at least 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

Certain embodiments provide a method of reducing HBV DNA and HBV antigen in a animal infected with a hepatitis B virus, comprising administering to the animal a compound or composition described herein. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as serum HBeAg absence plus serum HBeAb presence if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.

Certain embodiments provide a method for treating an animal with a HBV related disease, disorder or condition comprising: a) identifying said animal with the HBV related disease, disorder or condition, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363, as measured over the entirety of said modified oligonucleotide. In certain embodiments, the therapeutically effective amount of the compound or composition administered to the animal treats or reduces the HBV related disease, disorder or condition, or a symptom thereof, in the animal. In certain embodiments, the HBV related disease, disorder or condition is a liver disease. In certain embodiments, the related disease, disorder or condition is chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or liver disease-related to transplantation.

Certain embodiments provide a method for treating an animal with a HBV related disease, disorder or condition comprising: a) identifying said animal with the HBV related disease, disorder or condition, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to SEQ ID NO: 1, as measured over the entirety of said modified oligonucleotide. In certain embodiments, the therapeutically effective amount of the compound or composition administered to the animal treats or reduces the HBV related disease, disorder or condition, or a symptom thereof, in the animal. In certain embodiments, the HBV related disease, disorder or condition is a liver disease. In certain embodiments, the related disease, disorder or condition is chronic HBV infection, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or liver disease-related to transplantation.

In certain embodiments, HBV has the sequence as set forth in GenBank Accession Numbers U95551.1 (incorporated herein as SEQ ID NO: 1) or any variant or fragment thereof. In certain embodiments, HBV has truncated portions of the human sequence as set forth in SEQ ID NOs: 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1287, 1352, 1353, 1354, 1359, 1360, 1361, 1362, and 1363.

In certain embodiments, the animal is a human.

In certain embodiments, the compounds or compositions are designated as a first agent. In certain embodiments, the methods comprise administering a first agent and one or more second agents. In certain embodiments, the methods comprise administering a first agent and one or more second agents. In certain embodiments, the first agent and one or more second agents are co-administered. In certain embodiments the first agent and one or more second agents are co-administered sequentially or concomitantly.

In certain embodiments, the one or more second agents are also a compound or composition described herein. In certain embodiments, the one or more second agents are different from a compound or composition described herein. Examples of one or more second agents include, but are not limited to, an anti-inflammatory agent, chemotherapeutic agent or anti-infection agent.

In other related embodiments, the additional therapeutic agent may be an HBV agent, an HCV agent, a chemotherapeutic agent, an antibiotic, an analgesic, a non-steroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an anti-nausea agent, an anti-diarrheal agent, or an immunosuppressant agent.

In certain embodiments, the one or more second agents are an HBV agent. In certain embodiments the HBV agent can include, but is not limited to, interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; a second antisense oligomer; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or an HBV antibody therapy (monoclonal or polyclonal).

In certain embodiments, the one or more second agents are an HCV agent. In certain embodiments the HBV agent can include, but is not limited to interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV antisense agent; an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; or an HCV monoclonal or polyclonal antibody therapy.

In certain embodiments, the one or more second agents are an anti-inflammatory agent (i.e., an inflammation lowering therapy). In certain embodiments the inflammation lowering therapy can include, but is not limited to, a therapeutic lifestyle change, a steroid, a NSAID or a DMARD. The steroid can be a corticosteroid. The NSAID can be an aspirin, acetaminophen, ibuprofen, naproxen, COX inhibitors, indomethacin and the like. The DMARD can be a TNF inhibitor, purine synthesis inhibitor, calcineurin inhibitor, pyrimidine synthesis inhibitor, a sulfasalazine, methotrexate and the like.

In certain embodiments, the one or more second agents are a chemotherapeutic agent (i.e., a cancer treating agent). Chemotherapeutic agents can include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin, gemcitabine and diethylstilbestrol (DES).

In certain embodiments, the one or more second agents are an anti-infection agent. Examples of anti-infection agents include, but are not limited to, antibiotics, antifungal drugs and antiviral drugs.

In certain embodiments, administration comprises parenteral administration.

Certain embodiment provides a method for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment. In some embodiments, the mammal may be human, and the hepatitis B virus may be a human hepatitis B virus. More particularly, the human hepatitis B virus may be any of the human geographical genotypes: A (Northwest Europe, North America, Central America); B (Indonesia, China, Vietnam); C (East Asia, Korea, China, Japan, Polynesia, Vietnam); D (Mediterranean area, Middle East, India); E (Africa); F (Native Americans, Polynesia); G (United States, France); or H (Central America).

In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 70% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 75% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 80% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 85% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 90% compared to the amount before administration of the modified antisense oligonucleotide. In certain embodiments, a method is provided for reducing an amount of HBV mRNA, DNA, protein and/or an amount of HBV antigen in a mammal infected with a hepatitis B virus, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal in need thereof so as to reduce the hepatitis B virus infection and the hepatitis B antigen, compared to the amount of HBV mRNA, protein and an amount of HBV antigen in the mammal before treatment, wherein the amount of mRNA is reduced at least 95% compared to the amount before administration of the modified antisense oligonucleotide. In related methods, the HBV antigen may be HBsAg or may be HBeAg, and more particularly, the amount of HBV antigen may be sufficiently reduced to result in seroconversion, defined as serum HBeAg absence plus serum HBeAb presence if monitoring HBeAg as the determinant for seroconversion, or defined as serum HBsAg absence if monitoring HBsAg as the determinant for seroconversion, as determined by currently available detection limits of commercial ELISA systems.

Certain embodiment provides a method for promoting seroconversion of a hepatitis B virus in a mammal infected with HBV, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as described above to a mammal infected with hepatitis B; monitoring for presence of HBeAg plus HBeAb in a serum sample of the mammal, or monitoring for presence of HBsAg in a serum sample of the mammal, such that the absence of HBeAg plus the presence of HBeAb in the serum sample if monitoring HBeAg as the determinant for seroconversion, or the absence of HBsAg in the serum sample if monitoring HBsAg as the determinant for seroconversion, as determined by current detection limits of commercial ELISA systems, is indication of seroconversion in the mammal.

Certain embodiments provide the use of a compound or composition as described herein for preventing, ameliorating or treating liver disease, or symptom thereof, in an animal. In certain embodiments, the compound or composition comprises a modified oligonucleotide 10 to 30 linked nucleosides in length targeted to HBV. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17, 51, 86, 93, 95, 98, 100, 102, 104, 106, 109, 112, 115, 117, 137, 140, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 167, 168, 176, 177-179, 181, 188, 190-192, 194, 199, 201, 208, 209, 211, 226, 230-237, 244, 245, 247, 252, 254, 256, 258, 260, 262, 264, 266, 271, 1318-1347, 1364-1372, 1375, 1376, and 1379.

In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC₅₀ of less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55 nM, less than 50 nM, less than 49 nM, less than 47 nM, less than 46 nM, when delivered to HepG2.2.1 cells. In certain embodiments inhibition is measured with primer probe set RTS3370, as described herein.

In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC₅₀ of less than 250 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 35 nM, less than 34 nM, less than 33 nM, less than 32 nM, less than 31 nM, when delivered to HepG2.2.1 cells. In certain embodiments inhibition is measured with primer probe set RTS3371, as described herein.

In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC₅₀ of less than 20 μM, less than 10 μM, less than 9.5 μM, less than 9.0 μM, less than 8.5 μM, less than 8.0 μM, less than 7.5 μM, less than 7.0 μM, less than 6.5 μM, less than 6.0 μM, less than 5.5 μM, less than 5.0 μM, less than 4.5 μM, less than 4.0 μM, less than 3.5 μM, less than 3.0 μM, less than 2.5 μM, when delivered to HepG2.2.1 cells as described herein.

In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having at least one of an increase an ALT or AST value of no more than 4 fold, 3 fold, or 2 fold over saline treated animals or an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5%, or 2%. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase of ALT or AST over saline treated animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase in liver, spleen, or kidney weight over saline treated animals. In certain embodiments, these compounds or compositions include ISIS 146779, ISIS 146786, ISIS 505317, ISIS 505329, ISIS 505332, ISIS 505346, ISIS 505347, ISIS 505358, ISIS 509926, ISIS 509927, ISIS 509932, ISIS 509934, ISIS 509960, ISIS 509974, ISIS 510038, ISIS 510039, ISIS 510040, ISIS 510041, ISIS 510050 ISIS 509975, ISIS 510100, ISIS 510106, and ISIS 510116. In certain embodiments, such compounds or compositions include compounds comprising the nucleobase sequence of any one of SEQ ID NOs: 5-310, 321-802, or 804-1272. In certain embodiments, such compounds or compositions include compounds comprising the nucleobase sequence of any one of SEQ ID NOs: 5, 15, 16, 33, 39-95, 123-135, 163-175, 180-310, 321-406, 413-455, 461-802, or 804-1272. In certain embodiments, such compounds or compositions include compounds comprising the nucleobase sequence of any one of SEQ ID NOs: 6-14, 17-32, 34-38, 96-122, 136-162, 176-179, 407-412, 456-462, 523-538 (update SEQ ID NOs) wherein at least one nucleoside of the modified oligonucleotide comprises at least one 2′-O-methoxyethyl sugar. In certain embodiments, the modified oligonucleotide is 14 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-3 or 2 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 16 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 17 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 2-4 or 3-4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 18 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, 3-5, or 4 sugar modified nucleosides. In certain embodiments, the modified oligonucleotide is 20 nucleosides in length and has a gap segment of 10 linked nucleosides. In certain embodiments, the modified oligonucleotide has a wing segment on the 5′ end and 3′ end of the gap each independently having 1-5, or 5 sugar modified nucleosides.

Certain embodiments provide the use of a compound or composition as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing an HBV-related disease, disorder or condition in an animal.

Certain embodiments provide the use of a compound or composition as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing liver disease in an animal.

Certain embodiments provide a kit for treating, preventing, or ameliorating an HBV-related disease, disorder or condition, or a symptom thereof, as described herein wherein the kit comprises: a) a compound or compositions as described herein; and optionally b) an additional agent or therapy as described herein. The kit can further include instructions or a label for using the kit to treat, prevent, or ameliorate the HBV-related disease, disorder or condition.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 10-30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 12 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 12 to 22 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 14 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 14 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 15 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 15 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 16 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 16 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 17 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 17 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 to 30 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 to 21 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 to 20 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 14 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 16 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 17 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 18 subunits in length. In certain embodiments, an antisense compound targeted to a HBV nucleic acid is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.

In certain embodiments antisense oligonucleotides targeted to a HBV nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a HBV nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a HBV nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

In certain embodiments, gapmers provided herein include, for example, 11-mers having a motif of 1-9-1.

In certain embodiments, gapmers provided herein include, for example, 12-mers having a motif of 1-9-2, 2-9-1, or 1-10-1.

In certain embodiments, gapmers provided herein include, for example, 13-mers having a motif of 1-9-3, 2-9-2, 3-9-1, 1-10-2, or 2-10-1.

In certain embodiments, gapmers provided herein include, for example, 14-mers having a motif of 1-9-4, 2-9-3, 3-9-2, 4-9-1, 1-10-3, 2-10-2, or 3-10-1.

In certain embodiments, gapmers provided herein include, for example, 15-mers having a motif of 1-9-5, 2-9-4, 3-9-3, 4-9-2, 5-9-1, 1-10-4, 2-10-3, 3-10-2, or 4-10-1.

In certain embodiments, gapmers provided herein include, for example, 16-mers having a motif of 4-8-4, 2-9-5, 3-9-4, 4-9-3, 5-9-2, 1-10-5, 2-10-4, 3-10-3, 4-10-2, 3-8-5, or 5-10-1.

In certain embodiments, gapmers provided herein include, for example, 17-mers having a motif of 3-9-5, 3-10-4, 4-9-4, 5-9-3, 2-10-5, 3-10-4, 4-10-3, 5-10-2, 2-9-6, 5-8-4, 5-7-5, 6-7-4, or 6-9-2.

In certain embodiments, gapmers provided herein include, for example, 18-mers having a motif of 4-9-5, 5-9-4, 3-10-5, 4-10-4, or 5-10-3.

In certain embodiments, gapmers provided herein include, for example, 19-mers having a motif of 5-9-5, 4-10-5, or 5-10-4.

In certain embodiments, gapmers provided herein include, for example, 20-mers having a motif of 5-10-5, 2-10-8, 8-10-2, 3-10-7, 7-10-3, 4-10-6, or 6-10-4.

In certain embodiments, the antisense compound has a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration.

Thus, wingmer configurations provided herein include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-10-2 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-10-3 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-10-4 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 5-10-5 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-10-4 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-10-4 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-10-8 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 8-10-2 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-10-7 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 7-10-3 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-10-6 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 6-10-4 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-9-6 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 6-9-2 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-9-4 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 5-9-3 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-9-5 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 5-9-2 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 2-9-5 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 4-9-3 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a 3-9-4 gapmer motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a gap-widened motif.

In certain embodiments, the antisense compound targeted to a HBV nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense compounds targeted to a HBV nucleic acid has any of the following sugar motifs:

k-d(10)-k

e-d(10)-k

k-d(10)-e

k-k-d(10)-k-k

k-k-d(10)-e-e

e-e-d(10)-k-k

k-k-k-d(10)-k-k-k

e-e-e-d(10)-k-k-k

k-k-k-d(10)-e-e-e

k-k-k-d(10)-k-k-k

e-k-k-d(10)-k-k-e

e-e-k-d(10)-k-k-e

e-d-k-d(10)-k-k-e

e-k-d(10)-k-e-k-e

k-d(10)-k-e-k-e-e

e-e-k-d(10)-k-e-k-e

e-d-d-k-d(9)-k-k-e

e-e-e-e-d(9)-k-k-e

e-e-e-e-e-d(10)-e-e-e-e-e

k-d-k-d-k-d(9)-e-e

e-e-k-k-d(9)-e-k-e-e

k-d-k-d-k-d(10)-e-e-e-e-e

k-e-k-d(10)-k-e-k

e-e-e-k-k-d(8)-e-e-e-e

e-e-e-k-k-d(7)-k-k-e-e-e

e-e-e-k-d(9)-k-e-e-e

e-e-e-k-k-d(7)-k-k-e-e-e

e-e-e-e-k-k-d(7)-e-e-e-e

e-k-e-k-d(9)-e-e-e-e

e-k-e-k-d-k-d(7)-e-e-e-e

e-e-e-k-k-d(7)-k-k-e-e-e

k-d-k-d-k-d(8)-e-e-e-e-e

wherein, k is a constrained ethyl nucleoside, e is a 2′-MOE substituted nucleoside, and d is a 2′-deoxynucleoside.

In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode HBV include, without limitation, the following: GENBANK Accession U95551.1 (incorporated herein as SEQ ID NO: 1).

It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for HBV can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or stop codon.

The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in HBV mRNA levels are indicative of inhibition of HBV expression. Reductions in levels of a HBV protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes are indicative of inhibition of HBV expression. In certain embodiments, reduced fatigue, reduced flu-like symptoms, increase in appetite, reduced nausea, reduced joint pain, reduced jaundice, reduced pain in the abdomen, reduced weakness, reduced weight loss, reduction in breast enlargement in men, reduced rash on the palms, reduced difficulty with blood clotting, reduced cirrhosis, reduced spider-like blood vessels on the skin, increased Vitamins A and D absorption, reduced tumor growth, reduced tumor volume, reduced headache, reduced fever, reduced diarrhea, reduced pain over the liver area of the body, reduced clay- or grey-colored stool, reduced itching, reduced dark-colored urine, and reduced nausea and vomiting can be indicative of inhibition of HBV expression, In certain embodiments, amelioration of symptoms associated with HBV-related conditions, disease, and disorders can be indicative of inhibition of HBV expression. In certain embodiments, reduction of cirrhosis is indicative of inhibition of HBV expression. In certain embodiments, reduction of liver cancer markers can be indicative of inhibition of HBV expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a HBV nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a HBV nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a HBV nucleic acid).

Non-complementary nucleobases between an antisense compound and a HBV nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a HBV nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a HBV nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a HBV nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a HBV nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a HBV nucleic acid, or specified portion thereof.

The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a HBV nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

Antisense compounds provided herein can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups); bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C₁-C₁₂ alkyl or a protecting group); and combinations thereof. Examples of chemically modified sugars include, 2′-F-5′-methyl substituted nucleoside (see, PCT International Application WO 2008/101157, published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides), replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see, published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005), or, alternatively, 5′-substitution of a BNA (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH₃, and 2′-O(CH₂)2OCH₃ substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include, without limitation, nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4′ to 2′ bicyclic nucleoside. Examples of such 4′ to 2′ bicyclic nucleosides, include, but are not limited to, one of the formulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′, and analogs thereof (see, published PCT International Application WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′, and analogs thereof (see, published PCT International Application WO2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see, published U.S. Patent Application US2004/0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′, and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008). Also see, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,670,461, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 7,399,845; published PCT International applications WO 2004/106356, WO 94/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; and U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Application Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl, or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is, —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′,4′-(CH₂)_(3-2′, 4)′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH₂—O-2′) BNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH2-N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is the base moiety and R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleoside having Formula I:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—, —CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O—, or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium.

In certain embodiments, bicyclic nucleoside having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio.

In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃, OC(═X)J_(c), and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d), and J_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O or NJ_(c).

In certain embodiments, bicyclic nucleoside having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, or substituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleoside having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl, substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl, or substituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleoside having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl, or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (see, e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA, methyleneoxy (4′-CH₂—O-2′) BNA, and 2′-thio-BNAs, have also been prepared (see, e.g., Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (see, e.g., Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog, has been described in the art (see, e.g., Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleoside having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k), or N(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl, or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃₋₂′ bridge and the alkenyl analog, bridge 4′-CH═CH—CH₂-2′, have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

As used herein, “bicyclic nucleoside” refers to a nucleoside comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic sugar moiety. In certain embodiments, the bridge connects the 2′ carbon and another carbon of the sugar ring.

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting the 2′ carbon atom and the 4′ carbon atom.

As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)NH₂, O(CH₂)CH₃, O(CH₂)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)CH₃]₂, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂ alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl; aralkyl; O-alkaryl or O-aralkyl; SH; SCH₃; OCN; Cl; Br; CN; CF₃; OCF₃; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving pharmacokinetic properties; and a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (see, e.g., Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (see, e.g., Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F—HNA), or those compounds having Formula X:

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S, or NJ₁, and each J₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula X are provided wherein q_(m), q_(n), q_(p), q_(r), q_(s), q_(t), and q_(u) are each H. In certain embodiments, at least one of q_(m), q_(n), q_(p), q_(r), q_(s), q_(t), and q_(u) is other than H. In certain embodiments, at least one of q_(m), q_(n), q_(p), q_(r), q_(s), q_(t) and q_(u) is methyl. In certain embodiments, THP nucleosides of Formula X are provided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

As used herein, “2′-modified nucleoside” or “2′-substituted nucleoside” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position of a furanose ring other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R), or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. 2′-modified nucleosides may further comprise other modifications, for example, at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a sugar comprising a fluoro group at the 2′ position.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to those skilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleotides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a cEt. In certain embodiments, the cEt modified nucleotides are arranged throughout the wings of a gapmer motif.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

An antisense compound targeted to a HBV nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a HBV nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expression of HBV nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HuVEC cells, b.END cells, HepG2 cells, Hep3B cells, and primary hepatocytes.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a HBV nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents may be obtained from Invitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.

Probes and primers are designed to hybridize to a HBV nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).

Quantitative Real-Time PCR Analysis of Target DNA Levels

Quantitation of target DNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.

Gene (or DNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total DNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total DNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of DNA quantification by RIBOGREEN are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.

Probes and primers are designed to hybridize to a HBV nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of HBV nucleic acids can be assessed by measuring HBV protein levels.

Protein levels of HBV can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of HBV and produce phenotypic changes. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, subcutaneous, intrathecal, and intracerebroventricular. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from liver tissue and changes in HBV nucleic acid expression are measured. Changes in HBV DNA levels are also measured. Changes in HBV protein levels are also measured. Changes in HBV HBeAg levels are also measured. Changes in HBV HBsAg levels are also measured.

Certain Indications

In certain embodiments, provided herein are methods, compounds, and compositions of treating an individual comprising administering one or more pharmaceutical compositions provided herein. In certain embodiments, the individual has an HBV-related condition. In certain embodiments, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia. In certain embodiments, the HBV-related condition may have which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when coupled with a positive test for presence of a hepatitis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen. In certain embodiments, the individual is at risk for an HBV-related condition. This includes individuals having one or more risk factors for developing an HBV-related condition, including sexual exposure to an individual infected with Hepatitis B virus, living in the same house as an individual with a lifelong hepatitis B virus infection, exposure to human blood infected with the hepatitis B virus, injection of illicit drugs, being a person who has hemophilia, and visiting an area where hepatitis B is common. In certain embodiments, the individual has been identified as in need of treatment for an HBV-related condition. In certain embodiments provided herein are methods for prophylactically reducing HBV expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid.

Due to overlapping transmission routes, many people have been exposed to both hepatitis B virus (HBV) and hepatitis C virus (HCV), and a smaller proportion are chronically infected with both viruses, especially in regions such as Asia where HBV is endemic. Estimates suggest that up to 10% of people with HCV may also have HBV, while perhaps 20% of people with HBV are co-infected with HCV. However, treatment of hepatitis B or hepatitis B in HBV-HCV co-infected individuals has not been well studied. Treatment is complicated by the fact that HCV and HBV appear to inhibit each other's replication (though not all studied have observed this interaction). Therefore, treatment that fully suppresses HBV could potentially allow HCV to re-emerge, or vice versa. Therefore, the compounds and compositions described herein may advantageously be used for treating patients infected with both HBV and HCV. Exemplary treatment options for hepatitis C(HCV) include interferons, e.g., interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1. Less frequent interferon dosing can be achieved using pegylated interferon (interferon attached to a polyethylene glycol moiety which improves its pharmacokinetic profile). Combination therapy with interferon alpha-2b (pegylated and unpegylated) and ribavirin has also been shown to be efficacious for some patient populations. Other agents currently being developed include HCV RNA replication inhibitors (e.g., ViroPharma's VP50406 series), HCV antisense agents, HCV therapeutic vaccines, HCV protease inhibitors, HCV helicase inhibitors and HCV antibody therapy (monoclonal or polyclonal).

In certain embodiments, treatment with the methods, compounds, and compositions described herein is useful for preventing an HBV-related condition associated with the presence of the hepatitis B virus. In certain embodiments, treatment with the methods, compounds, and compositions described herein is useful for preventing an HBV-related condition.

In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV mRNA levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV DNA levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV protein levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV S antigen (HBsAg) levels in the serum of an individual to determine an individual's response to administration of the antisense compound. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an HBV nucleic acid is accompanied by monitoring of HBV E antigen (HBeAg) levels in the serum of an individual to determine an individual's response to administration of the antisense compound. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.

In certain embodiments, administration of an antisense compound targeted to an HBV nucleic acid results in reduction of HBV expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to an HBV nucleic acid results in reduced symptoms associated with the HBV-related condition and reduced HBV-related markers in the blood. In certain embodiments, administration of an HBV antisense compound decreases HBV RNA levels, HBV DNA levels, HBV protein levels, HBsAg levels, or HBeAg levels by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to HBV are used for the preparation of a medicament for treating a patient suffering or susceptible to an HBV-related condition.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions provided herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions provided herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions provided herein. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions provided herein are co-administered with another pharmaceutical agent to produce a synergistic effect.

In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions provided herein and one or more other pharmaceutical agents are prepared separately. In certain embodiments the antisense oligonucleotides disclosed is administered in combination with an HCV agent. In further embodiments, the HCV compound is administered simultaneously as the antisense compound; in other embodiments, the HCV compound is administered separately; so that a dose of each of the HCV agent and the antisense compound overlap, in time, within the patient's body. In related embodiments, the HCV agent may be selected from interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated); ribavirin; an HCV RNA replication inhibitor (e.g., ViroPharma's VP50406 series); an HCV antisense agent; an HCV therapeutic vaccine; an HCV protease inhibitor; an HCV helicase inhibitor; and an HCV antibody therapy (monoclonal or polyclonal).

In other embodiments, an HBV antisense compound of the present invention may be administered to a patient infected with HBV, in combination with one or more HBV therapeutic agents, wherein the one or more HBV therapeutic agents may be administered in the same drug formulation as the HBV ASO compound, or may be administered in a separate formulation. The one or more HBV therapeutic agents may be administered simultaneously with the ASO HBV compound, or may be administered separately, so that a dose of each of the HBV ASO compound and the HBV therapeutic agent overlap, in time, within the patient's body. In related embodiments, the one or more HBV therapeutic agent may be selected from interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and unpegylated), ribavirin; an HBV RNA replication inhibitor; a second HBV antisense compound; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir; tenofovir; telbivudine (LdT); adefovir; and an HBV antibody therapy (monoclonal or polyclonal).

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

Example 1 Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Cultured HepG2.2.15 cells at a density of 25,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 2; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 3; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 4) was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in Table 1 were designed as either 5-10-5 MOE gapmers or 3-10-4 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and 4 nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. The ‘Motif’ column indicates the gap and wing structure of each gapmer. Each gapmer listed in Table 1 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).

TABLE 1 Inhibition of viral HBV mRNA levels by MOE gapmers targeted to SEQ ID NO: 1 Viral Viral Start Stop % SEQ ID Site Site ISIS No Sequence Motif inhibition NO 245 261 510088 CCACGAGTCTAGACTCT 3-10-4 55 5 250 266 510089 GTCCACCACGAGTCTAG 3-10-4 59 6 251 267 510090 AGTCCACCACGAGTCTA 3-10-4 60 7 252 268 510091 AAGTCCACCACGAGTCT 3-10-4 47 8 253 269 510092 GAAGTCCACCACGAGTC 3-10-4 59 9 254 270 510093 AGAAGTCCACCACGAGT 3-10-4 32 10 255 271 510094 GAGAAGTCCACCACGAG 3-10-4 41 11 256 272 510095 AGAGAAGTCCACCACGA 3-10-4 44 12 257 273 510096 GAGAGAAGTCCACCACG 3-10-4 54 13 258 274 510097 TGAGAGAAGTCCACCAC 3-10-4 57 14 384 400 510098 TGATAAAACGCCGCAGA 3-10-4 55 15 385 401 510099 ATGATAAAACGCCGCAG 3-10-4 59 16 411 427 510100 GGCATAGCAGCAGGATG 3-10-4 85 17 412 428 510101 AGGCATAGCAGCAGGAT 3-10-4 51 18 413 429 510102 GAGGCATAGCAGCAGGA 3-10-4 69 19 414 433 505330 AGATGAGGCATAGCAGCAGG 5-10-5 74 20 414 430 510103 TGAGGCATAGCAGCAGG 3-10-4 12 21 415 434 509928 AAGATGAGGCATAGCAGCAG 5-10-5 71 22 415 431 510104 ATGAGGCATAGCAGCAG 3-10-4 69 23 416 435 509929 GAAGATGAGGCATAGCAGCA 5-10-5 78 24 416 432 510105 GATGAGGCATAGCAGCA 3-10-4 69 25 417 436 509930 AGAAGATGAGGCATAGCAGC 5-10-5 72 26 417 433 510106 AGATGAGGCATAGCAGC 3-10-4 77 27 418 437 146783 AAGAAGATGAGGCATAGCAG 5-10-5 15 28 418 434 510107 AAGATGAGGCATAGCAG 3-10-4 69 29 419 435 510108 GAAGATGAGGCATAGCA 3-10-4 59 30 420 436 510109 AGAAGATGAGGCATAGC 3-10-4 0 31 421 437 510110 AAGAAGATGAGGCATAG 3-10-4 38 32 457 473 510111 ACGGGCAACATACCTTG 3-10-4 62 33 639 658 146784 CTGAGGCCCACTCCCATAGG 5-10-5 5 34 639 655 510112 AGGCCCACTCCCATAGG 3-10-4 44 35 640 656 510113 GAGGCCCACTCCCATAG 3-10-4 27 36 641 657 510114 TGAGGCCCACTCCCATA 3-10-4 44 37 642 658 510115 CTGAGGCCCACTCCCAT 3-10-4 52 38 687 706 509931 CGAACCACTGAACAAATGGC 5-10-5 89 39 687 703 510116 ACCACTGAACAAATGGC 3-10-4 89 40 688 704 510117 AACCACTGAACAAATGG 3-10-4 69 41 689 705 510118 GAACCACTGAACAAATG 3-10-4 63 42 690 706 510119 CGAACCACTGAACAAAT 3-10-4 74 43 738 754 510120 ACCACATCATCCATATA 3-10-4 71 44 1176 1192 510121 TCAGCAAACACTTGGCA 3-10-4 73 45 1778 1797 509932 AATTTATGCCTACAGCCTCC 5-10-5 76 46 1778 1794 510122 TTATGCCTACAGCCTCC 3-10-4 76 47 1779 1798 509933 CAATTTATGCCTACAGCCTC 5-10-5 72 48 1779 1795 510123 TTTATGCCTACAGCCTC 3-10-4 75 49 1780 1799 509934 CCAATTTATGCCTACAGCCT 5-10-5 75 50 1780 1796 510124 ATTTATGCCTACAGCCT 3-10-4 73 51 1781 1800 509935 ACCAATTTATGCCTACAGCC 5-10-5 72 52 1781 1797 510125 AATTTATGCCTACAGCC 3-10-4 69 53 1782 1798 510126 CAATTTATGCCTACAGC 3-10-4 59 54 1783 1799 510127 CCAATTTATGCCTACAG 3-10-4 58 55 1784 1800 510128 ACCAATTTATGCCTACA 3-10-4 60 56 1822 1838 510129 AGGCAGAGGTGAAAAAG 3-10-4 47 57 1823 1839 510130 TAGGCAGAGGTGAAAAA 3-10-4 30 58 1865 1884 509936 GCACAGCTTGGAGGCTTGAA 5-10-5 39 59 1865 1881 510131 CAGCTTGGAGGCTTGAA 3-10-4 4 60 1866 1885 509937 GGCACAGCTTGGAGGCTTGA 5-10-5 35 61 1866 1882 510132 ACAGCTTGGAGGCTTGA 3-10-4 0 62 1867 1886 505370 AGGCACAGCTTGGAGGCTTG 5-10-5 36 63 1867 1883 510133 CACAGCTTGGAGGCTTG 3-10-4 12 64 1868 1887 509938 AAGGCACAGCTTGGAGGCTT 5-10-5 7 65 1868 1884 510134 GCACAGCTTGGAGGCTT 3-10-4 20 66 1869 1888 509939 CAAGGCACAGCTTGGAGGCT 5-10-5 36 67 1869 1885 510135 GGCACAGCTTGGAGGCT 3-10-4 22 68 1870 1889 505371 CCAAGGCACAGCTTGGAGGC 5-10-5 35 69 1870 1886 510136 AGGCACAGCTTGGAGGC 3-10-4 14 70 1871 1887 510137 AAGGCACAGCTTGGAGG 3-10-4 0 71 1872 1888 510138 CAAGGCACAGCTTGGAG 3-10-4 6 72 1873 1889 510139 CCAAGGCACAGCTTGGA 3-10-4 17 73 1918 1934 510140 GCTCCAAATTCTTTATA 3-10-4 59 74 2378 2397 509940 TCTGCGAGGCGAGGGAGTTC 3-10-4 10 75 2378 2394 510141 GCGAGGCGAGGGAGTTC 3-10-4 5 76 2379 2395 510142 TGCGAGGCGAGGGAGTT 3-10-4 0 77 2380 2396 510143 CTGCGAGGCGAGGGAGT 3-10-4 8 78 2381 2397 510144 TCTGCGAGGCGAGGGAG 3-10-4 17 79 2820 2836 510145 TTCCCAAGAATATGGTG 3-10-4 22 80 2821 2837 510146 GTTCCCAAGAATATGGT 3-10-4 11 81 2822 2838 510147 TGTTCCCAAGAATATGG 3-10-4 21 82

Example 2 Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Cultured HepG2.2.15 cells at a density of 25,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. RTS3370 detects the full length mRNA and the second portions of the pre-S1, pre-S2 and pre-C mRNA transcripts. The gapmers were also probed with additional primer probe sets. Viral primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 311; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 312; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 313) was used also to measure mRNA levels. RTS3371 detects the full length mRNA and the second portions of the pre-S1, pre-S2 and pre-C mRNA transcripts, similar to RTS3370, but at different regions. Viral primer probe set RTS3372 (forward sequence ATCCTATCAACACTTCCGGAAACT, designated herein as SEQ ID NO: 314; reverse sequence CGACGCGGCGATTGAG, designated herein as SEQ ID NO: 315; probe sequence AAGAACTCCCTCGCCTCGCAGACG, designated herein as SEQ ID NO: 316) was used to measure mRNA levels. RTS3372 detects the full length genomic sequence. Viral primer probe set RTS3373MGB (forward sequence CCGACCTTGAGGCATACTTCA, designated herein as SEQ ID NO: 317; reverse sequence AATTTATGCCTACAGCCTCCTAGTACA, designated herein as SEQ ID NO: 318; probe sequence TTAAAGACTGGGAGGAGTTG, designated herein as SEQ ID NO: 319) was used to measure mRNA levels. RTS3373MGB detects the full length mRNA and the second portions of the pre-S1, pre-S2, pre-C, and pre-X mRNA transcripts.

HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in Table 2 were designed as either 5-10-5 MOE gapmers, 3-10-3 MOE gapmers, or 2-10-2 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The 3-10-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleosides each. The 2-10-2 MOE gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.

“Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. The ‘Motif’ column indicates the gap and wing structure of each gapmer. Each gapmer listed in Table 2 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).

TABLE 2 Inhibition of viral HBV mRNA levels by MOE gapmers targeted to SEQ ID NO: 1 (detected by RTS3370, RTS3371, RTS3372, and RTS3373MGB) RTS3373 SEQ Start Stop RTS3370 % RTS3371 % RTS3372 % MGB % ID Site Site ISIS No Sequence inhibition inhibition inhibition inhibition Motif NO 58 77 146779 GAACTGGA 76 80 82 81 5-10-5 83 GCCACCAG CAGG 58 71 510019 GAGCCACC 38 32 45 31 2-10-2 84 AGCAGG 61 80 505314 CCTGAACT 68 71 67 66 5-10-5 85 GGAGCCAC CAGC 62 77 509941 GAACTGGA 36 32 71 53 3-10-3 86 GCCACCAG 196 215 505315 AAAAACCC 69 74 80 88 5-10-5 87 CGCCTGTA ACAC 199 218 505316 AAGAAAAA 60 60 64 64 5-10-5 88 CCCCGCCTG TAA 205 224 505317 GTCAACAA 85 83 79 85 5-10-5 89 GAAAAACC CCGC 228 241 510020 GTATTGTGA 28 18 0 16 2-10-2 90 GGATT 229 242 510021 GGTATTGTG 40 37 19 34 2-10-2 91 AGGAT 244 263 146821 CACCACGA 74 73 62 75 5-10-5 92 GTCTAGACT CTG 245 260 509942 CACGAGTC 18 15 45 46 3-10-3 93 TAGACTCT 245 258 510022 CGAGTCTA 32 26 23 19 2-10-2 94 GACTCT 246 261 509943 CCACGAGT 34 35 63 60 3-10-3 95 CTAGACTC 247 266 505318 GTCCACCA 75 77 64 75 5-10-5 96 CGAGTCTA GACT 250 269 509921 GAAGTCCA 46 46 39 40 5-10-5 97 CCACGAGT CTAG 250 265 509944 TCCACCAC 38 39 65 59 3-10-3 98 GAGTCTAG 251 270 509922 AGAAGTCC 55 56 17 38 5-10-5 99 ACCACGAG TCTA 251 266 509945 GTCCACCA 34 35 64 51 3-10-3 100 CGAGTCTA 252 271 509923 GAGAAGTC 39 38 39 33 5-10-5 101 CACCACGA GTCT 252 267 509946 AGTCCACC 47 51 50 45 3-10-3 102 ACGAGTCT 253 272 505319 AGAGAAGT 88 83 80 78 5-10-5 103 CCACCACG AGTC 253 268 509947 AAGTCCAC 46 50 56 46 3-10-3 104 CACGAGTC 254 273 509924 GAGAGAAG 43 40 49 44 5-10-5 105 TCCACCAC GAGT 254 269 509948 GAAGTCCA 41 46 51 44 3-10-3 106 CCACGAGT 254 267 510023 AGTCCACC 41 32 47 48 2-10-2 107 ACGAGT 255 274 509925 TGAGAGAA 50 57 55 55 5-10-5 108 GTCCACCA CGAG 255 270 509949 AGAAGTCC 40 41 52 34 3-10-3 109 ACCACGAG 255 268 510024 AAGTCCAC 26 29 19 23 2-10-2 110 CACGAG 256 275 505320 TTGAGAGA 51 57 55 66 5-10-5 111 AGTCCACC ACGA 256 271 509950 GAGAAGTC 30 31 43 33 3-10-3 112 CACCACGA 256 269 510025 GAAGTCCA 44 38 53 54 2-10-2 113 CCACGA 257 270 510026 AGAAGTCC 39 42 32 25 2-10-2 114 ACCACG 258 273 509952 GAGAGAAG 54 52 60 48 3-10-3 115 TCCACCAC 258 271 510027 GAGAAGTC 29 30 25 19 2-10-2 116 CACCAC 259 274 509953 TGAGAGAA 39 44 47 38 3-10-3 117 GTCCACCA 259 272 510028 AGAGAAGT 31 29 3 15 2-10-2 118 CCACCA 260 273 510029 GAGAGAAG 21 19 23 18 2-10-2 119 TCCACC 261 274 510030 TGAGAGAA 16 22 21 20 2-10-2 120 GTCCAC 262 281 505321 AGAAAATT 53 58 52 56 5-10-5 121 GAGAGAAG TCCA 265 284 505322 CCTAGAAA 62 65 69 67 5-10-5 122 ATTGAGAG AAGT 293 312 505323 ATTTTGGCC 86 84 81 85 5-10-5 123 AAGACACA CGG 296 315 505324 CGAATTTTG 67 67 69 64 5-10-5 124 GCCAAGAC ACA 302 321 505325 GGACTGCG 77 75 73 76 5-10-5 125 AATTTTGGC CAA 360 379 505326 TCCAGCGA 89 90 77 91 5-10-5 126 TAACCAGG ACAA 366 385 505327 GACACATC 83 85 75 86 5-10-5 127 CAGCGATA ACCA 369 388 505328 GCAGACAC 65 68 49 57 5-10-5 128 ATCCAGCG ATAA 384 399 509954 GATAAAAC 37 46 53 35 3-10-3 129 GCCGCAGA 384 397 510031 TAAAACGC 36 36 33 33 2-10-2 130 CGCAGA 385 398 510032 ATAAAACG 12 7 19 15 2-10-2 131 CCGCAG 386 401 509955 ATGATAAA 49 55 57 53 3-10-3 132 ACGCCGCA 386 399 510033 GATAAAAC 39 39 45 37 2-10-2 133 GCCGCA 387 400 510034 TGATAAAA 40 37 29 39 2-10-2 134 CGCCGC 388 401 510035 ATGATAAA 22 24 9 22 2-10-2 135 ACGCCG 411 430 505329 TGAGGCAT 60 64 47 55 5-10-5 136 AGCAGCAG GATG 411 426 509956 GCATAGCA 62 64 71 60 3-10-3 137 GCAGGATG 411 424 510036 ATAGCAGC 44 34 30 48 2-10-2 138 AGGATG 412 431 509926 ATGAGGCA 45 54 71 62 5-10-5 139 TAGCAGCA GGAT 412 427 509957 GGCATAGC 72 75 80 71 3-10-3 140 AGCAGGAT 412 425 510037 CATAGCAG 29 24 24 20 2-10-2 141 CAGGAT 413 432 509927 GATGAGGC 54 58 54 49 5-10-5 142 ATAGCAGC AGGA 413 428 509958 AGGCATAG 63 66 68 64 3-10-3 143 CAGCAGGA 413 426 510038 GCATAGCA 55 54 37 46 2-10-2 144 GCAGGA 414 433 505330 AGATGAGG 85 87 74 82 5-10-5 20 CATAGCAG CAGG 414 429 509959 GAGGCATA 64 64 80 68 3-10-3 145 GCAGCAGG 414 427 510039 GGCATAGC 58 54 41 45 2-10-2 146 AGCAGG 415 430 509960 TGAGGCAT 59 59 66 64 3-10-3 147 AGCAGCAG 415 428 510040 AGGCATAG 58 55 38 41 2-10-2 148 CAGCAG 416 431 509961 ATGAGGCA 56 54 65 56 3-10-3 149 TAGCAGCA 416 429 510041 GAGGCATA 64 62 64 57 2-10-2 150 GCAGCA 417 432 509962 GATGAGGC 57 52 58 49 3-10-3 151 ATAGCAGC 417 430 510042 TGAGGCAT 48 50 55 48 2-10-2 152 AGCAGC 418 433 509963 AGATGAGG 50 52 64 51 3-10-3 153 CATAGCAG 418 431 510043 ATGAGGCA 36 31 36 26 2-10-2 154 TAGCAG 419 434 509964 AAGATGAG 48 47 72 65 3-10-3 155 GCATAGCA 419 432 510044 GATGAGGC 44 28 0 14 2-10-2 156 ATAGCA 420 435 509965 GAAGATGA 45 41 65 62 3-10-3 157 GGCATAGC 420 433 510045 AGATGAGG 41 43 37 29 2-10-2 158 CATAGC 421 436 509966 AGAAGATG 32 29 64 51 3-10-3 159 AGGCATAG 421 434 510046 AAGATGAG 21 18 26 27 2-10-2 160 GCATAG 422 437 509967 AAGAAGAT 21 17 55 46 3-10-3 161 GAGGCATA 422 435 510047 GAAGATGA 25 24 23 25 2-10-2 162 GGCATA 423 436 510048 AGAAGATG 21 17 25 19 2-10-2 163 AGGCAT 424 437 510049 AAGAAGAT 17 11 38 27 2-10-2 164 GAGGCA 454 473 505331 ACGGGCAA 55 57 65 60 5-10-5 165 CATACCTTG ATA 457 476 505332 CAAACGGG 73 77 77 74 5-10-5 166 CAACATAC CTTG 457 472 509968 CGGGCAAC 60 61 73 70 3-10-3 167 ATACCTTG 458 473 509969 ACGGGCAA 58 63 64 58 3-10-3 168 CATACCTT 458 471 510050 GGGCAACA 58 56 57 46 2-10-2 169 TACCTT 459 472 510051 CGGGCAAC 49 43 47 37 2-10-2 170 ATACCT 460 473 510052 ACGGGCAA 50 50 54 51 2-10-2 171 CATACC 463 482 505333 AGAGGACA 64 68 64 71 5-10-5 172 AACGGGCA ACAT 466 485 505334 ATTAGAGG 59 62 42 69 5-10-5 173 ACAAACGG GCAA 472 491 505335 CCTGGAATT 78 81 73 86 5-10-5 174 AGAGGACA AAC 475 494 505336 GATCCTGG 56 65 61 72 5-10-5 175 AATTAGAG GACA 639 654 509970 GGCCCACT 38 55 74 48 3-10-3 176 CCCATAGG 641 656 509971 GAGGCCCA 30 46 77 54 3-10-3 177 CTCCCATA 642 657 509972 TGAGGCCC 58 57 84 66 3-10-3 178 ACTCCCAT 643 658 509973 CTGAGGCC 38 53 70 66 3-10-3 179 CACTCCCA 670 689 146823 GGCACTAG 61 64 63 63 5-10-5 180 TAAACTGA GCCA 670 685 509974 CTAGTAAA 71 71 78 80 3-10-3 181 CTGAGCCA 670 683 510053 AGTAAACT 49 48 52 53 2-10-2 182 GAGCCA 671 684 510054 TAGTAAAC 41 38 19 30 2-10-2 183 TGAGCC 672 685 510055 CTAGTAAA 25 27 42 47 2-10-2 184 CTGAGC 673 692 505337 AATGGCAC 34 46 49 52 5-10-5 185 TAGTAAAC TGAG 679 698 505338 TGAACAAA 74 77 71 80 5-10-5 186 TGGCACTA GTAA 682 701 505339 CACTGAAC 82 83 71 82 5-10-5 187 AAATGGCA CTAG 687 702 509975 CCACTGAA 72 73 76 80 3-10-3 188 CAAATGGC 688 707 505340 ACGAACCA 69 69 78 76 5-10-5 189 CTGAACAA ATGG 688 703 509976 ACCACTGA 47 48 67 65 3-10-3 190 ACAAATGG 689 704 509977 AACCACTG 33 33 39 41 3-10-3 191 AACAAATG 690 705 509978 GAACCACT 50 49 63 48 3-10-3 192 GAACAAAT 691 710 505341 CCTACGAA 64 70 70 72 5-10-5 193 CCACTGAA CAAA 691 706 509979 CGAACCAC 67 66 78 77 3-10-3 194 TGAACAAA 691 704 510056 AACCACTG 36 36 23 32 2-10-2 195 AACAAA 692 705 510057 GAACCACT 45 44 51 43 2-10-2 196 GAACAA 693 706 510058 CGAACCAC 59 52 48 49 2-10-2 197 TGAACA 697 716 505342 GAAAGCCC 76 80 73 83 5-10-5 198 TACGAACC ACTG 738 753 509980 CCACATCAT 40 33 62 54 3-10-3 199 CCATATA 738 751 510059 ACATCATCC 19 9 30 27 2-10-2 200 ATATA 739 754 509981 ACCACATC 76 78 93 85 3-10-3 201 ATCCATAT 739 752 510060 CACATCATC 45 35 24 17 2-10-2 202 CATAT 740 753 510061 CCACATCAT 52 49 43 40 2-10-2 203 CCATA 741 754 510062 ACCACATC 44 45 48 47 2-10-2 204 ATCCAT 756 775 505343 TGTACAGA 47 56 55 68 5-10-5 205 CTTGGCCCC CAA 823 842 505344 AGGGTTTA 66 71 64 72 5-10-5 206 AATGTATA CCCA 1170 1189 505345 GCAAACAC 76 80 35 70 5-10-5 207 TTGGCACA GACC 1176 1191 509982 CAGCAAAC 42 44 56 54 3-10-3 208 ACTTGGCA 1177 1192 509983 TCAGCAAA 60 54 74 70 3-10-3 209 CACTTGGC 1259 1278 505346 CCGCAGTA 88 82 57 80 5-10-5 210 TGGATCGG CAGA 1261 1276 509984 GCAGTATG 61 58 65 72 3-10-3 211 GATCGGCA 1262 1281 505347 GTTCCGCA 84 81 71 83 5-10-5 212 GTATGGAT CGGC 1268 1287 505348 CTAGGAGT 78 68 70 79 5-10-5 213 TCCGCAGT ATGG 1271 1290 505349 CGGCTAGG 47 54 59 61 5-10-5 214 AGTTCCGC AGTA 1277 1296 505350 AACAAGCG 55 62 69 69 5-10-5 215 GCTAGGAG TTCC 1280 1299 505351 CAAAACAA 20 49 49 54 5-10-5 216 GCGGCTAG GAGT 1283 1302 505352 GAGCAAAA 53 83 73 87 5-10-5 217 CAAGCGGC TAGG 1286 1305 505353 TGCGAGCA 64 73 68 78 5-10-5 218 AAACAAGC GGCT 1413 1426 510063 ACAAAGGA 14 8 0 0 2-10-2 219 CGTCCC 1515 1534 505354 GAGGTGCG 68 81 61 80 5-10-5 220 CCCCGTGGT CGG 1518 1537 505355 AGAGAGGT 59 75 75 84 5-10-5 221 GCGCCCCG TGGT 1521 1540 505356 TAAAGAGA 63 76 83 78 5-10-5 222 GGTGCGCC CCGT 1550 1563 510064 AAGGCACA 35 38 25 32 2-10-2 223 GACGGG 1577 1596 146786 GTGAAGCG 88 91 84 93 5-10-5 224 AAGTGCAC ACGG 1580 1599 505357 GAGGTGAA 70 75 71 82 5-10-5 225 GCGAAGTG CACA 1583 1602 505358 GCAGAGGT 77 82 72 84 5-10-5 226 GAAGCGAA GTGC 1586 1605 505359 CGTGCAGA 72 73 67 80 5-10-5 227 GGTGAAGC GAAG 1655 1674 505360 AGTCCAAG 66 68 54 68 5-10-5 228 AGTCCTCTT ATG 1706 1719 510065 CAGTCTTTG 19 19 26 17 2-10-2 229 AAGTA 1778 1793 509985 TATGCCTAC 64 60 64 63 3-10-3 230 AGCCTCC 1779 1794 509986 TTATGCCTA 66 66 77 73 3-10-3 231 CAGCCTC 1780 1795 509987 TTTATGCCT 56 55 68 67 3-10-3 232 ACAGCCT 1781 1796 509988 ATTTATGCC 52 52 68 63 3-10-3 233 TACAGCC 1782 1797 509989 AATTTATGC 48 44 70 59 3-10-3 234 CTACAGC 1783 1798 509990 CAATTTATG 24 18 39 40 3-10-3 235 CCTACAG 1784 1799 509991 CCAATTTAT 37 37 55 55 3-10-3 236 GCCTACA 1785 1800 509992 ACCAATTTA 35 36 60 55 3-10-3 237 TGCCTAC 1806 1825 505361 AAAGTTGC 42 55 75 61 5-10-5 238 ATGGTGCT GGTG 1809 1828 505362 GAAAAAGT 45 56 64 53 5-10-5 239 TGCATGGT GCTG 1812 1831 505363 GGTGAAAA 71 70 80 72 5-10-5 240 AGTTGCAT GGTG 1815 1834 505364 AGAGGTGA 51 57 77 82 5-10-5 241 AAAAGTTG CATG 1818 1837 505365 GGCAGAGG 54 63 76 78 5-10-5 242 TGAAAAAG TTGC 1821 1840 505366 TTAGGCAG 61 65 80 66 5-10-5 243 AGGTGAAA AAGT 1822 1837 509993 GGCAGAGG 47 51 74 54 3-10-3 244 TGAAAAAG 1823 1838 509994 AGGCAGAG 47 40 76 54 3-10-3 245 GTGAAAAA 1824 1843 505367 TGATTAGG 41 39 62 29 5-10-5 246 CAGAGGTG AAAA 1824 1839 509995 TAGGCAGA 46 42 79 59 3-10-3 247 GGTGAAAA 1826 1839 510066 TAGGCAGA 40 33 44 31 2-10-2 248 GGTGAA 1827 1846 505368 AGATGATT 27 46 62 51 5-10-5 249 AGGCAGAG GTGA 1861 1880 146787 AGCTTGGA 59 61 65 72 5-10-5 250 GGCTTGAA CAGT 1864 1883 505369 CACAGCTT 11 21 48 31 5-10-5 251 GGAGGCTT GAAC 1865 1880 509996 AGCTTGGA 13 1 45 40 3-10-3 252 GGCTTGAA 1865 1878 510067 CTTGGAGG 22 17 20 14 2-10-2 253 CTTGAA 1866 1881 509997 CAGCTTGG 29 19 51 45 3-10-3 254 AGGCTTGA 1866 1879 510068 GCTTGGAG 24 25 37 32 2-10-2 255 GCTTGA 1867 1886 505370 AGGCACAG 32 36 58 33 5-10-5 63 CTTGGAGG CTTG 1867 1882 509998 ACAGCTTG 1 4 23 12 3-10-3 256 GAGGCTTG 1867 1880 510069 AGCTTGGA 23 24 17 23 2-10-2 257 GGCTTG 1868 1883 509999 CACAGCTT 5 1 48 41 3-10-3 258 GGAGGCTT 1868 1881 510070 CAGCTTGG 21 20 0 18 2-10-2 259 AGGCTT 1869 1884 510000 GCACAGCT 14 10 50 37 3-10-3 260 TGGAGGCT 1869 1882 510071 ACAGCTTG 19 22 24 27 2-10-2 261 GAGGCT 1870 1889 505371 CCAAGGCA 27 40 68 38 5-10-5 69 CAGCTTGG AGGC 1870 1885 510001 GGCACAGC 10 12 43 16 3-10-3 262 TTGGAGGC 1870 1883 510072 CACAGCTT 28 31 33 30 2-10-2 263 GGAGGC 1871 1886 510002 AGGCACAG 24 20 46 25 3-10-3 264 CTTGGAGG 1871 1884 510073 GCACAGCT 20 18 22 15 2-10-2 265 TGGAGG 1872 1887 510003 AAGGCACA 6 0 45 24 3-10-3 266 GCTTGGAG 1872 1885 510074 GGCACAGC 18 18 32 23 2-10-2 267 TTGGAG 1873 1892 505372 CACCCAAG 18 8 55 16 5-10-5 268 GCACAGCT TGGA 1873 1888 510004 CAAGGCAC 9 0 31 15 3-10-3 269 AGCTTGGA 1873 1886 510075 AGGCACAG 23 9 27 10 2-10-2 270 CTTGGA 1874 1889 510005 CCAAGGCA 0 0 39 25 3-10-3 271 CAGCTTGG 1876 1895 505373 AGCCACCC 47 50 69 56 5-10-5 272 AAGGCACA GCTT 1879 1898 505374 CAAAGCCA 27 27 55 30 5-10-5 273 CCCAAGGC ACAG 1882 1901 505375 CCCCAAAG 34 40 54 39 5-10-5 274 CCACCCAA GGCA 1885 1904 505376 ATGCCCCA 41 43 54 52 5-10-5 275 AAGCCACC CAAG 1888 1907 505377 TCCATGCCC 40 42 72 40 5-10-5 276 CAAAGCCA CCC 1891 1910 505378 ATGTCCATG 35 33 70 40 5-10-5 277 CCCCAAAG CCA 1918 1933 510006 CTCCAAATT 9 2 53 41 3-10-3 278 CTTTATA 1918 1931 510076 CCAAATTCT 28 22 7 22 2-10-2 279 TTATA 1919 1934 510007 GCTCCAAA 43 39 72 57 3-10-3 280 TTCTTTAT 1919 1932 510077 TCCAAATTC 19 11 0 2 2-10-2 281 TTTAT 1920 1933 510078 CTCCAAATT 19 11 0 0 2-10-2 282 CTTTA 1921 1934 510079 GCTCCAAA 50 48 61 55 2-10-2 283 TTCTTT 1957 1976 505379 GGAAAGAA 17 14 81 39 5-10-5 284 GTCAGAAG GCAA 2270 2285 510008 GTGCGAAT 21 4 36 11 3-10-3 285 CCACACTC 2270 2283 510080 GCGAATCC 32 29 41 33 2-10-2 286 ACACTC 2271 2284 510081 TGCGAATC 28 20 25 11 2-10-2 287 CACACT 2272 2285 510082 GTGCGAAT 28 20 32 22 2-10-2 288 CCACAC 2368 2387 505380 GAGGGAGT 24 22 90 48 5-10-5 289 TCTTCTTCT AGG 2378 2393 510009 CGAGGCGA 12 1 65 10 3-10-3 290 GGGAGTTC 2378 2391 510083 AGGCGAGG 17 18 29 25 2-10-2 291 GAGTTC 2379 2394 510010 GCGAGGCG 18 13 82 37 3-10-3 292 AGGGAGTT 2379 2392 510084 GAGGCGAG 29 22 54 30 2-10-2 293 GGAGTT 2380 2395 510011 TGCGAGGC 13 11 69 44 3-10-3 294 GAGGGAGT 2380 2393 510085 CGAGGCGA 25 20 53 42 2-10-2 295 GGGAGT 2381 2396 510012 CTGCGAGG 17 14 79 53 3-10-3 296 CGAGGGAG 2381 2394 510086 GCGAGGCG 33 29 66 48 2-10-2 297 AGGGAG 2382 2397 510013 TCTGCGAG 18 4 77 47 3-10-3 298 GCGAGGGA 2420 2439 505381 CCGAGATT 12 18 83 28 5-10-5 299 GAGATCTTC TGC 2459 2478 505382 CCCACCTTA 14 19 80 36 5-10-5 300 TGAGTCCA AGG 2819 2838 505383 TGTTCCCAA 29 32 78 44 5-10-5 301 GAATATGG TGA 2820 2835 510014 TCCCAAGA 10 10 68 40 3-10-3 302 ATATGGTG 2821 2836 510015 TTCCCAAG 5 0 62 24 3-10-3 303 AATATGGT 2822 2837 510016 GTTCCCAA 6 2 42 16 3-10-3 304 GAATATGG 2823 2838 510017 TGTTCCCAA 18 18 47 18 3-10-3 305 GAATATG 2824 2839 510018 TTGTTCCCA 7 5 57 19 3-10-3 306 AGAATAT 2825 2838 510087 TGTTCCCAA 25 20 44 25 2-10-2 307 GAATA 2873 2892 505384 GAAAGAAT 8 4 61 22 5-10-5 308 CCCAGAGG ATTG 3161 3180 146833 ACTGCATG 47 46 82 54 5-10-5 309 GCCTGAGG ATGA 3163 3182 505385 CCACTGCAT 25 34 69 19 5-10-5 310 GGCCTGAG GAT

Example 3 Antisense Inhibition of HBV Viral mRNA in HepAD38 (Tet-HBV) Cells by MOE Gapmers

Certain antisense oligonucleotides selected from the study described in Example 2 were tested for their effects on HBV mRNA in another cell line, human hepatoma HepAD38 cells, in which HBV production is under the control of a tetracycline-regulated promoter. Cultured HepAD38 (Tet-HBV) cells at a density of 45,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe sets RTS3372 and RTS3373MGB were used individually to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 3 as percent inhibition of HBV, relative to untreated control cells.

TABLE 3 Inhibition of viral HBV mRNA levels by MOE gapmersin HepAD38 (Tet-HBV) cells (detected by RTS3372 and RTS3373MGB) Start Stop ISIS RTS3373MGB RTS3372 SEQ Site Site No Motif % inhibition % inhibition ID NO 58 77 146779 5-10-5 76 82 83 58 71 510019 5-10-5 0 9 84 61 80 505314 5-10-5 65 75 85 196 215 505315 5-10-5 46 65 87 199 218 505316 5-10-5 57 71 88 205 224 505317 5-10-5 83 87 89 228 241 510020 2-10-2 6 0 90 229 242 510021 2-10-2 19 24 91 244 263 146821 5-10-5 72 71 92 245 258 510022 2-10-2 6 24 94 247 266 505318 5-10-5 68 77 96 250 269 509921 5-10-5 25 47 97 251 270 509922 5-10-5 28 46 99 252 271 509923 5-10-5 19 40 101 253 272 505319 5-10-5 69 66 103 254 273 509924 5-10-5 9 39 105 254 267 510023 2-10-2 19 15 107 255 274 509925 5-10-5 26 55 108 255 268 510024 2-10-2 0 5 110 256 275 505320 5-10-5 62 68 111 256 269 510025 2-10-2 0 8 113 257 270 510026 2-10-2 7 21 114 258 271 510027 2-10-2 0 0 116 259 272 510028 2-10-2 0 0 118 260 273 510029 2-10-2 0 9 119 261 274 510030 2-10-2 0 0 120 262 281 505321 5-10-5 53 54 121 265 284 505322 5-10-5 59 60 122 293 312 505323 5-10-5 65 77 123 296 315 505324 5-10-5 78 83 124 302 321 505325 5-10-5 71 80 125 360 379 505326 5-10-5 76 84 126 366 385 505327 5-10-5 77 83 127 369 388 505328 5-10-5 65 78 128 384 397 510031 2-10-2 0 16 130 385 398 510032 2-10-2 0 0 131 386 399 510033 2-10-2 1 21 133 387 400 510034 2-10-2 8 28 134 388 401 510035 2-10-2 0 0 135 411 430 505329 5-10-5 58 72 136 411 424 510036 2-10-2 6 11 138 412 431 509926 5-10-5 20 54 139 412 425 510037 2-10-2 0 10 141 413 432 509927 5-10-5 56 76 142 413 426 510038 2-10-2 54 68 144 414 433 505330 5-10-5 66 81 20 414 427 510039 2-10-2 60 74 146 415 428 510040 2-10-2 33 39 148 416 429 510041 2-10-2 30 58 150 417 430 510042 2-10-2 34 57 152 418 431 510043 2-10-2 0 2 154 419 432 510044 2-10-2 0 29 156 420 433 510045 2-10-2 3 31 158 421 434 510046 2-10-2 0 0 160 422 435 510047 2-10-2 0 0 162 423 436 510048 2-10-2 0 0 163 424 437 510049 2-10-2 0 0 164 454 473 505331 5-10-5 60 77 165 457 476 505332 5-10-5 55 74 166 458 471 510050 2-10-2 47 47 169 459 472 510051 2-10-2 35 55 170 460 473 510052 2-10-2 27 41 171 463 482 505333 5-10-5 66 78 172 466 485 505334 5-10-5 53 63 173 472 491 505335 5-10-5 70 76 174 475 494 505336 5-10-5 64 77 175 670 689 146823 5-10-5 74 79 180 670 683 510053 2-10-2 18 20 182 671 684 510054 2-10-2 13 21 183 672 685 510055 2-10-2 4 2 184 673 692 505337 5-10-5 60 72 185 679 698 505338 5-10-5 62 75 186 682 701 505339 5-10-5 81 90 187 688 707 505340 5-10-5 67 81 189 691 710 505341 5-10-5 68 80 193 691 704 510056 2-10-2 0 0 195 692 705 510057 2-10-2 37 48 196 693 706 510058 2-10-2 44 59 197 697 716 505342 5-10-5 80 87 198 738 751 510059 2-10-2 0 0 200 739 752 510060 2-10-2 0 0 202 740 753 510061 2-10-2 23 19 203 741 754 510062 2-10-2 25 30 204 756 775 505343 5-10-5 62 71 205 823 842 505344 5-10-5 52 66 206 1170 1189 505345 5-10-5 83 81 207 1259 1278 505346 5-10-5 84 81 210 1262 1281 505347 5-10-5 89 84 212 1268 1287 505348 5-10-5 78 78 213 1271 1290 505349 5-10-5 74 77 214 1277 1296 505350 5-10-5 75 77 215 1280 1299 505351 5-10-5 49 62 216 1283 1302 505352 5-10-5 70 66 217 1286 1305 505353 5-10-5 62 60 218 1413 1426 510063 2-10-2 0 0 219 1515 1534 505354 5-10-5 85 75 220 1518 1537 505355 5-10-5 81 74 221 1521 1540 505356 5-10-5 57 52 222 1550 1563 510064 2-10-2 0 0 223 1577 1596 146786 5-10-5 94 85 224 1580 1599 505357 5-10-5 86 79 225 1583 1602 505358 5-10-5 89 79 226 1586 1605 505359 5-10-5 82 68 227 1655 1674 505360 5-10-5 84 74 228 1706 1719 510065 2-10-2 0 0 229 1806 1825 505361 5-10-5 66 66 238 1809 1828 505362 5-10-5 52 59 239 1812 1831 505363 5-10-5 72 75 240 1815 1834 505364 5-10-5 73 80 241 1818 1837 505365 5-10-5 68 82 242 1821 1840 505366 5-10-5 50 76 243 1824 1843 505367 5-10-5 58 76 246 1826 1839 510066 2-10-2 0 31 248 1827 1846 505368 5-10-5 71 84 249 1861 1880 146787 5-10-5 25 35 250 1864 1883 505369 5-10-5 29 65 251 1865 1878 510067 2-10-2 0 0 253 1866 1879 510068 2-10-2 0 20 255 1867 1886 505370 5-10-5 45 70 63 1867 1880 510069 2-10-2 0 0 257 1868 1881 510070 2-10-2 0 0 259 1869 1882 510071 2-10-2 0 0 261 1870 1889 505371 5-10-5 48 66 69 1870 1883 510072 2-10-2 0 0 263 1871 1884 510073 2-10-2 0 0 265 1872 1885 510074 2-10-2 0 2 267 1873 1892 505372 5-10-5 48 67 268 1873 1886 510075 2-10-2 0 0 270 1876 1895 505373 5-10-5 23 48 272 1879 1898 505374 5-10-5 0 34 273 1882 1901 505375 5-10-5 39 66 274 1885 1904 505376 5-10-5 0 40 275 1888 1907 505377 5-10-5 4 47 276 1891 1910 505378 5-10-5 65 77 277 1918 1931 510076 2-10-2 0 0 279 1919 1932 510077 2-10-2 0 0 281 1920 1933 510078 2-10-2 0 0 282 1921 1934 510079 2-10-2 18 50 283 1957 1976 505379 5-10-5 42 84 284 2270 2283 510080 2-10-2 0 0 286 2271 2284 510081 2-10-2 0 0 287 2272 2285 510082 2-10-2 0 10 288 2368 2387 505380 5-10-5 29 79 289 2378 2391 510083 2-10-2 0 0 291 2379 2392 510084 2-10-2 31 17 293 2380 2393 510085 2-10-2 0 8 295 2381 2394 510086 2-10-2 10 2 297 2420 2439 505381 5-10-5 30 86 299 2459 2478 505382 5-10-5 16 87 300 2819 2838 505383 5-10-5 26 81 301 2825 2838 510087 2-10-2 0 0 307 2873 2892 505384 5-10-5 31 59 308 3161 3180 146833 5-10-5 55 76 309 3163 3182 505385 5-10-5 58 83 310

Example 4 Antisense Inhibition of HBV Viral mRNA in HepAD38 (Tet-HBV) Cells by MOE Gapmers

Certain antisense oligonucleotides from the study described in Examples 1 and 2 were tested for their effects on HBV mRNA in vitro. Cultured HepAD38 (Tet-HBV) cells at a density of 45,000 cells per well were transfected using electroporation with 15,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3372 was used to measure mRNA levels. The mRNA levels were also measured using the RTS3373MGB primer probe set. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 4 as percent inhibition of HBV, relative to untreated control cells.

TABLE 4 Inhibition of viral HBV mRNA levels by MOE gapmers (RTS3372 and RTS3373MGB) Start Stop ISIS RTS3372 RTS3373MGB SEQ Site Site No Motif % inhibition % inhibition ID NO 62 77 509941 3-10-3 36 5 86 245 260 509942 3-10-3 3 0 93 245 261 510088 3-10-4 24 10 5 246 261 509943 3-10-3 27 13 95 250 265 509944 3-10-3 46 34 98 250 266 510089 3-10-4 61 33 6 251 266 509945 3-10-3 54 43 100 251 267 510090 3-10-4 58 32 7 252 267 509946 3-10-3 50 28 102 252 268 510091 3-10-4 60 42 8 253 268 509947 3-10-3 49 40 104 253 269 510092 3-10-4 40 9 9 254 269 509948 3-10-3 13 22 106 254 270 510093 3-10-4 39 2 10 255 270 509949 3-10-3 33 24 109 255 271 510094 3-10-4 40 16 11 256 271 509950 3-10-3 31 23 112 256 272 510095 3-10-4 24 6 12 257 273 510096 3-10-4 62 44 13 258 273 509952 3-10-3 42 40 115 258 274 510097 3-10-4 65 48 14 259 274 509953 3-10-3 35 29 117 384 399 509954 3-10-3 35 18 129 384 400 510098 3-10-4 62 43 15 385 401 510099 3-10-4 67 50 16 386 401 509955 3-10-3 44 37 132 411 426 509956 3-10-3 67 53 137 411 427 510100 3-10-4 88 69 17 412 427 509957 3-10-3 86 76 140 412 428 510101 3-10-4 71 46 18 413 428 509958 3-10-3 78 74 143 413 429 510102 3-10-4 77 52 19 414 433 505330 5-10-5 81 60 20 414 429 509959 3-10-3 62 49 145 414 430 510103 3-10-4 9 5 21 415 434 509928 5-10-5 81 66 22 415 430 509960 3-10-3 67 57 147 415 431 510104 3-10-4 71 57 23 416 435 509929 5-10-5 82 69 24 416 431 509961 3-10-3 62 43 149 416 432 510105 3-10-4 81 64 25 417 436 509930 5-10-5 74 45 26 417 432 509962 3-10-3 59 48 151 417 433 510106 3-10-4 86 70 27 418 437 146783 5-10-5 19 3 28 418 433 509963 3-10-3 48 28 153 418 434 510107 3-10-4 74 51 29 419 434 509964 3-10-3 50 39 155 419 435 510108 3-10-4 67 50 30 420 435 509965 3-10-3 49 38 157 420 436 510109 3-10-4 12 13 31 421 436 509966 3-10-3 23 22 159 421 437 510110 3-10-4 34 16 32 422 437 509967 3-10-3 3 12 161 457 472 509968 3-10-3 56 38 167 457 473 510111 3-10-4 68 51 33 458 473 509969 3-10-3 53 39 168 639 658 146784 5-10-5 0 0 34 639 654 509970 3-10-3 51 15 176 639 655 510112 3-10-4 66 32 35 640 656 510113 3-10-4 70 31 36 641 656 509971 3-10-3 54 31 177 641 657 510114 3-10-4 67 45 37 642 657 509972 3-10-3 51 25 178 642 658 510115 3-10-4 73 50 38 643 658 509973 3-10-3 49 32 179 670 685 509974 3-10-3 74 67 181 687 706 509931 5-10-5 92 83 39 687 702 509975 3-10-3 72 71 188 687 703 510116 3-10-4 83 74 40 688 703 509976 3-10-3 46 52 190 688 704 510117 3-10-4 71 57 41 689 704 509977 3-10-3 18 22 191 689 705 510118 3-10-4 71 50 42 690 705 509978 3-10-3 57 37 192 690 706 510119 3-10-4 80 64 43 691 706 509979 3-10-3 65 55 194 738 753 509980 3-10-3 48 44 199 738 754 510120 3-10-4 70 54 44 739 754 509981 3-10-3 54 45 201 1176 1191 509982 3-10-3 44 36 208 1176 1192 510121 3-10-4 74 69 45 1177 1192 509983 3-10-3 57 53 209 1261 1276 509984 3-10-3 57 50 211 1778 1797 509932 5-10-5 30 76 46 1778 1793 509985 3-10-3 0 46 230 1778 1794 510122 3-10-4 0 60 47 1779 1798 509933 5-10-5 54 78 48 1779 1794 509986 3-10-3 56 81 231 1779 1795 510123 3-10-4 74 85 49 1780 1799 509934 5-10-5 69 84 50 1780 1795 509987 3-10-3 52 78 232 1780 1796 510124 3-10-4 75 84 51 1781 1800 509935 5-10-5 72 85 52 1781 1796 509988 3-10-3 57 68 232 1781 1797 510125 3-10-4 68 72 53 1782 1797 509989 3-10-3 46 41 234 1782 1798 510126 3-10-4 56 51 54 1783 1798 509990 3-10-3 16 25 234 1783 1799 510127 3-10-4 61 69 55 1784 1799 509991 3-10-3 41 41 236 1784 1800 510128 3-10-4 61 68 56 1785 1800 509992 3-10-3 43 43 237 1822 1837 509993 3-10-3 72 44 244 1822 1838 510129 3-10-4 66 33 57 1823 1838 509994 3-10-3 79 32 245 1823 1839 510130 3-10-4 49 31 58 1824 1839 509995 3-10-3 63 30 247 1865 1884 509936 5-10-5 74 59 59 1865 1880 509996 3-10-3 36 0 252 1865 1881 510131 3-10-4 26 0 60 1866 1885 509937 5-10-5 78 63 61 1866 1881 509997 3-10-3 5 0 254 1866 1882 510132 3-10-4 37 4 62 1867 1886 505370 5-10-5 54 17 63 1867 1882 509998 3-10-3 13 0 256 1867 1883 510133 3-10-4 42 25 64 1868 1887 509938 5-10-5 9 6 65 1868 1883 509999 3-10-3 47 6 258 1868 1884 510134 3-10-4 56 27 66 1869 1888 509939 5-10-5 64 29 67 1869 1884 510000 3-10-3 24 1 260 1869 1885 510135 3-10-4 70 43 68 1870 1889 505371 5-10-5 63 46 69 1870 1885 510001 3-10-3 39 12 262 1870 1886 510136 3-10-4 52 23 70 1871 1886 510002 3-10-3 10 0 264 1871 1887 510137 3-10-4 28 0 71 1872 1887 510003 3-10-3 21 0 266 1872 1888 510138 3-10-4 25 7 72 1873 1888 510004 3-10-3 21 38 269 1873 1889 510139 3-10-4 18 0 73 1874 1889 510005 3-10-3 8 0 271 1918 1933 510006 3-10-3 0 0 278 1918 1934 510140 3-10-4 81 67 74 1919 1934 510007 3-10-3 69 66 280 2270 2285 510008 3-10-3 23 0 285 2378 2397 509940 3-10-4 66 7 75 2378 2393 510009 3-10-3 23 0 290 2378 2394 510141 3-10-4 10 11 76 2379 2394 510010 3-10-3 39 6 292 2379 2395 510142 3-10-4 46 24 77 2380 2395 510011 3-10-3 33 23 294 2380 2396 510143 3-10-4 59 36 78 2381 2396 510012 3-10-3 38 22 296 2381 2397 510144 3-10-4 54 20 79 2382 2397 510013 3-10-3 42 0 298 2820 2835 510014 3-10-3 51 9 302 2820 2836 510145 3-10-4 68 19 80 2821 2836 510015 3-10-3 35 2 303 2821 2837 510146 3-10-4 65 15 81 2822 2837 510016 3-10-3 9 0 304 2822 2838 510147 3-10-4 30 0 85 2823 2838 510017 3-10-3 18 0 305 2824 2839 510018 3-10-3 24 5 306

Example 5 Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers

Certain gapmers from the study described in Examples 3 and 4 were tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 25,000 cells per well and transfected using electroporation with 2.5 μM, 5.0 μM, 10.0 μM, and 20.0 μM concentrations of antisense oligonucleotide, as specified in Table 5. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 5. As illustrated in Table 5, HBV mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 5 Dose-dependent antisense inhibition of HBV RNA in HepG2.2.15 cells using RTS3370 IC₅₀ ISIS No 2.5 μM 5.0 μM 10.0 μM 20.0 μM (μM) 146786 33 50 54 81 5.7 505317 35 40 63 67 6.6 505323 16 33 48 63 11.1 505326 27 44 64 67 6.9 509929 21 44 60 62 8.4 509931 51 63 75 75 <2.5 509957 37 53 57 70 5.4 509974 25 35 54 63 9.5 509975 36 55 62 81 4.7 509981 7 23 35 52 18.8 510039 27 46 60 69 6.9 510040 10 28 43 59 13.4 510041 29 41 53 66 8.3 510058 9 34 42 63 11.9

Example 6 Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers

Additional gapmers from the study described in Examples 3 and 4 were further tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE 2000® reagent with 15.625 nM, 31.25 nM, 62.5 nM, 125.0 nM, and 250.0 nM concentrations of antisense oligonucleotide, as specified in Table 6. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 6. As illustrated in Table 6, HBV mRNA levels were significantly reduced in a dose-dependent manner in some antisense oligonucleotide treated cells.

TABLE 6 Dose-dependent antisense inhibition of HBV RNA in HepG2.2.15 cells using RTS3370 ISIS 15.625 31.25 62.5 125.0 250.0 IC₅₀ No nM nM nM nM nM (nM) 146779 14 25 44 70 78 73.1 146786 10 35 64 85 93 49.4 146833 12 16 32 62 72 99.8 505317 19 31 44 69 83 65.2 505319 5 11 24 39 69 152.8 505323 2 11 26 68 90 85.4 505326 1 15 45 72 89 73.7 505327 0 4 12 56 74 128.5 505329 3 16 33 51 64 130.4 505339 26 32 59 82 92 46.0 505342 10 4 34 69 74 95.7 505347 20 26 41 70 92 63.0 505356 0 0 0 38 69 182.0 505358 8 28 47 71 84 67.9 505382 5 0 3 26 19 >250.0 509926 0 6 18 42 67 159.3 509927 3 17 33 55 76 103.2 509929 7 19 36 60 69 102.9 509931 18 28 52 76 87 57.4 509934 14 14 40 61 76 89.3 509957 20 28 51 71 79 63.1 509958 12 17 37 56 76 96.4 509959 12 11 18 59 70 121.7 509960 9 19 30 57 74 103.4 509972 15 6 17 27 45 >250.0 509974 25 35 57 83 92 45.3 509975 33 44 45 61 80 53.1 509981 0 15 11 35 60 224.4 510007 0 0 15 31 45 >250.0 510038 12 19 48 73 84 68.9 510039 17 25 44 69 72 77.3 510040 17 20 23 59 72 108.6 510041 11 21 43 64 79 80.5 510050 3 21 16 51 70 132.4 510058 7 9 16 22 46 >250.0 510079 0 6 11 29 32 >250.0 510100 18 34 50 79 83 56.1 510106 23 25 35 69 74 78.4 510116 20 44 65 79 91 42.6 510140 7 28 30 55 58 136.5

The mRNA levels were also measured with primer probe set RTS3371. The results are presented in Table 7.

TABLE 7 Dose-dependent antisense inhibition of HBV RNA in HepG2.2.15 cells using RTS3371 ISIS 15.625 31.25 62.5 125.0 250.0 IC₅₀ No nM nM nM nM nM (nM) 146779 16 7 38 69 68 96.9 146786 28 39 65 86 93 35 146833 26 22 52 61 65 82.3 505317 18 33 40 77 84 61.4 505319 0 0 0 15 55 >250.0 505323 0 0 33 66 87 100.5 505326 0 21 7 57 85 114.6 505327 0 0 40 50 63 132.3 505329 11 22 35 66 77 90.7 505342 15 0 1 40 59 190.1 505347 3 35 44 65 90 68.4 505356 0 0 3 42 76 153.2 505358 20 11 39 71 78 79.7 505382 0 0 0 0 0 >250.0 509926 0 4 14 55 72 130.6 509927 11 25 31 61 78 88.4 509929 11 26 41 70 77 75.8 509931 25 39 55 79 85 46.6 509934 0 25 32 54 65 119.9 509957 25 44 48 74 80 50.6 509958 24 18 20 57 72 114.5 509959 2 9 31 52 65 132.3 509960 16 28 22 57 75 101.8 509972 3 5 1 39 60 236.3 509974 38 46 65 83 94 31.2 509975 30 7 24 49 67 148.2 509981 22 22 23 46 58 194.7 510007 3 0 15 33 39 >250.0 510038 16 22 50 76 84 62.9 510039 23 36 32 70 68 79.7 510040 18 15 41 59 67 101.9 510041 0 27 38 62 81 84.5 510050 1 16 17 52 63 149 510058 20 19 40 44 51 214.1 510079 0 2 5 41 49 >250.0 510100 35 52 61 86 90 30.7 510106 27 23 5 75 81 87.9 510116 11 44 70 72 94 46.5 510140 0 18 26 45 41 >250.0

Example 7 Tolerability of MOE Gapmers Targeting HBV in BALB/c Mice

BALB/c mice (Charles River, Mass.) are a multipurpose model of mice, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various metabolic markers.

Study 1

Groups of four BALB/c mice each were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 146779, ISIS 146786, ISIS 505317, ISIS 505319, ISIS 505330, ISIS 505332, ISIS 505339, ISIS 505346, ISIS 505347, ISIS 505358, ISIS 509929, ISIS 509931, ISIS 509932, ISIS 509934, ISIS 509957, ISIS 510100, ISIS 510106, ISIS 510116, and ISIS 510140. A group of four BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 141923 (CCTTCCCTGAAGGTTCCTCC (SEQ ID NO: 320)), a 5-10-5 MOE gapmer with no known homology to any human or mouse gene sequence. Another group of 4 BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. This group of mice served as the control group. Three days after the last dose at each time point, body weights were taken, mice were euthanized and organs and plasma were harvested for further analysis.

Body and Organ Weights

The body weights of the mice were measured pre-dose and at the end of each treatment period. The body weights are presented in Table 8, and are expressed as percent change from the weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are presented in Table 9 as a percentage difference from the respective organ weights of the PBS control. The results indicate that most of the ISIS oligonucleotides did not cause any adverse effects on body or organ weights.

TABLE 8 Change in body weights of BALB/c mice after antisense oligonucleotide treatment (%) Body weight PBS 9 ISIS 141923 9 ISIS 146779 11 ISIS 146786 9 ISIS 505317 10 ISIS 505319 14 ISIS 505330 11 ISIS 505332 10 ISIS 505339 14 ISIS 505346 12 ISIS 505347 16 ISIS 505358 12 ISIS 509929 8 ISIS 509931 9 ISIS 509932 21 ISIS 509934 14 ISIS 509957 10 ISIS 510100 10 ISIS 510106 15 ISIS 510116 16 ISIS 510140 19

TABLE 9 Change in organ weights of BALB/c mice after antisense oligonucleotide treatment (%) Liver Kidney Spleen PBS — — — ISIS 141923 3 −3 −9 ISIS 146779 10 1 13 ISIS 146786 19 −3 4 ISIS 505317 −4 −7 9 ISIS 505319 1 −16 23 ISIS 505330 12 −4 9 ISIS 505332 7 −2 14 ISIS 505339 5 −6 7 ISIS 505346 7 −6 0 ISIS 505347 12 −7 5 ISIS 505358 8 0 3 ISIS 509929 17 14 200 ISIS 509931 −4 −9 3 ISIS 509932 18 −9 79 ISIS 509934 6 −6 2 ISIS 509957 0 −2 15 ISIS 510100 2 1 8 ISIS 510106 5 −2 58 ISIS 510116 12 −8 7 ISIS 510140 20 −8 49 Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 10 expressed in IU/L. Plasma levels of cholesterol and triglycerides were also measured using the same clinical chemistry analyzer and the results are also presented in Table 10.

TABLE 10 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of BALB/c mice ALT AST Cholesterol Triglycerides (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 37 58 114 238 ISIS 141923 36 57 114 234 ISIS 146779 43 56 121 221 ISIS 146786 53 76 118 327 ISIS 505317 68 103 117 206 ISIS 505319 136 152 144 168 ISIS 505330 281 194 119 188 ISIS 505332 67 70 123 226 ISIS 505339 113 111 135 249 ISIS 505346 56 63 128 234 ISIS 505347 79 83 122 347 ISIS 505358 78 175 112 214 ISIS 509929 111 166 61 175 ISIS 509931 635 508 110 179 ISIS 509932 92 113 118 131 ISIS 509934 38 89 97 176 ISIS 509957 159 229 85 173 ISIS 510100 90 87 86 222 ISIS 510106 61 88 79 239 ISIS 510116 70 95 124 214 ISIS 510140 1247 996 161 167 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 11, expressed in mg/dL.

TABLE 11 Effect of antisense oligonucleotide treatment on kidney markers of BALB/c mice BUN (mg/dL) PBS 29 ISIS 141923 29 ISIS 146779 28 ISIS 146786 30 ISIS 505317 30 ISIS 505319 30 ISIS 505330 29 ISIS 505332 28 ISIS 505339 29 ISIS 505346 27 ISIS 505347 26 ISIS 505358 26 ISIS 509929 25 ISIS 509931 23 ISIS 509932 28 ISIS 509934 25 ISIS 509957 24 ISIS 510100 27 ISIS 510106 27 ISIS 510116 25 ISIS 510140 22 Study 2

Groups of four BALB/c mice each were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 505329, ISIS 509926, ISIS 509927, ISIS 509958, ISIS 509959, ISIS 509960, ISIS 509974, ISIS 509975, ISIS 510038, ISIS 510039, ISIS 510040, ISIS 510041, and ISIS 510050. A group of 4 BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. This group of mice served as the control group. Three days after the last dose at each time point, body weights were taken, mice were euthanized and organs and plasma were harvested for further analysis.

Organ Weights

Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 12 as a percentage change over the respective organ weights of the PBS control.

TABLE 12 Change in organ weights of BALB/c mice after antisense oligonucleotide treatment (%) ISIS No Liver Kidney Spleen 505329 12 2 12 509926 23 3 30 509927 8 −4 27 509958 1 −4 9 509959 7 0 26 509960 16 6 30 509974 5 8 7 509975 1 −1 7 510038 6 4 23 510039 0 15 9 510040 3 1 2 510041 6 6 10 510050 5 5 18 Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 13 expressed in IU/L.

TABLE 13 Effect of antisense oligonucleotide treatment on transaminases (IU/L) in the liver of BALB/c mice ALT AST PBS 37 78 ISIS 505329 48 65 ISIS 509926 77 120 ISIS 509927 71 92 ISIS 509958 106 105 ISIS 509959 119 122 ISIS 509960 40 66 ISIS 509974 38 43 ISIS 509975 33 45 ISIS 510038 69 66 ISIS 510039 32 61 ISIS 510040 83 113 ISIS 510041 32 45 ISIS 510050 26 47 Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 14, expressed in mg/dL.

TABLE 14 Effect of antisense oligonucleotide treatment on kidney markers of BALB/c mice BUN PBS 21 ISIS 505329 22 ISIS 509926 20 ISIS 509927 20 ISIS 509958 22 ISIS 509959 21 ISIS 509960 20 ISIS 509974 19 ISIS 509975 19 ISIS 510038 19 ISIS 510039 19 ISIS 510040 22 ISIS 510041 18 ISIS 510050 22

Example 8 Dose Response Confirmation of MOE Gapmers Targeting HBV in HepG2.2.15 Cells

Gapmers were chosen based on sequence conservation, activity and tolerability, as measured in the study described in Examples 7 and 8, and tested at various doses in HepG2.2.15 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE 2000 reagent with 15.625 nM, 31.25 nM, 62.5 nM, 125.0 nM and 250.0 nM concentrations of antisense oligonucleotide. Two days post-transfection, the media was replaced with fresh media. Samples were collected 4 days post-transfection. DNA, RNA, HBsAg and HBeAg levels were measured in the supernatant.

HBV mRNA levels were measured by quantitative real-time PCR. HBV primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells. As illustrated in Table 15, HBV mRNA levels were reduced in a dose-dependent manner in most of the antisense oligonucleotide treated cells.

HBV antigens in the supernatants were detected with the ELISA technique. HBs antigen (HBsAg) levels were detected by ELISA from Abazyme LLC, MA. As presented in Table 16, treatment with ISIS oligonucleotides 146779, 146786, 505329, 505330, 505339, 505347, 505358, 509927, 509934, 509958, 509959, 509960, 509974, 5100038, 510039, 510040, 510041, 510100, 510106, and 510116 caused significant reduction in HBsAg levels. HBe antigen (HBeAg) levels were detected by ELISA from International Immuno-diagnostics, CA. As presented in Table 17, treatment with ISIS oligonucleotides 146779, 146786, 505329, 505330, 505339, 505347, 505358, 509927, 509934, 509958, 509959, 509960, 509974, 5100038, 510039, 510040, 510041, 510100, 510106, and 510116 caused significant reduction in HBeAg levels. HBV DNA levels were measured using primer probe set RTS3370. As presented in Table 18, treatment with ISIS oligonucleotides 146779, 146786, 505329, 505330, 505339, 505347, 505358, 509927, 509934, 509958, 509959, 509960, 509974, 5100038, 510039, 510040, 510041, 510100, 510106, and 510116 caused significant reduction in HBV DNA levels. The total protein in the supernatants was measured by a DC protein assay (BioRad), as presented in Table 19.

TABLE 15 Dose-dependent antisense inhibition of HBV RNA in HepG2.2.15cells ISIS No 15.625 nM 31.25 nM 62.5 nM 125 nM 250 nM 146779 10 25 42 64 95 146786 23 59 78 84 90 505329 45 49 57 69 83 505330 31 61 65 80 93 505339 31 56 78 89 97 505347 30 50 72 87 96 505358 28 52 75 86 95 509927 41 61 67 61 76 509934 38 61 64 82 58 509958 50 67 72 79 89 509959 50 63 73 80 86 509960 63 61 72 82 74 509974 29 44 75 91 96 510038 29 40 85 89 93 510039 32 34 63 84 84 510040 18 0 51 71 77 510041 34 53 67 76 71 510100 29 64 70 89 93 510106 28 65 64 81 85 510116 13 34 78 89 95

TABLE 16 Dose-dependent reduction of S antigen in HepG2.2.15 cell supernatant ISIS No 15.625 nM 31.25 nM 62.5 nM 125 nM 146779 40 58 80 92 146786 47 75 92 98 505329 37 58 71 89 505330 45 66 84 95 505339 62 79 93 96 505347 68 71 89 97 505358 69 83 92 96 509927 54 74 88 94 509934 40 59 78 89 509958 57 77 91 93 509959 54 72 84 100 509960 44 72 91 91 509974 58 77 92 95 510038 58 78 94 98 510039 53 74 89 95 510040 39 70 80 90 510041 47 65 82 92 510100 74 83 95 96 510106 54 75 86 92 510116 61 74 91 94

TABLE 17 Dose-dependent reduction of E antigen in HepG2.2.15 cell supernatant ISIS No 15.625 nM 31.25 nM 62.5 nM 125 nM 146779 14 45 66 76 146786 26 58 75 80 505329 19 26 60 73 505330 28 70 69 80 505339 31 57 77 82 505347 24 33 64 77 505358 26 45 72 81 509927 34 54 72 79 509934 21 42 59 73 509958 29 45 72 77 509959 60 64 77 80 509960 19 36 67 77 509974 16 48 72 80 510038 20 35 79 80 510039 14 41 64 78 510040 0 8 37 69 510041 9 34 63 76 510100 26 52 73 81 510106 7 42 62 76 510116 27 56 76 81

TABLE 18 Dose-dependent antisense inhibition of HBV DNA in HepG2.2.15 cells ISIS No 15.625 nM 31.25 nM 62.5 nM 125 nM 146779 71 71 84 85 146786 67 81 82 75 505329 53 65 72 67 505330 72 76 86 90 505339 83 85 89 88 505347 76 78 81 87 505358 79 82 90 87 509927 51 75 78 69 509934 61 60 64 75 509958 57 73 69 71 509959 59 54 73 73 509960 48 66 63 54 509974 76 90 84 85 510038 69 76 90 87 510039 70 79 81 86 510040 40 67 68 68 510041 53 71 62 68 510100 76 81 87 87 510106 46 74 73 76 510116 79 84 89 86

TABLE 19 Total protein levels in HepG2.2.15 cell supernatant 15.625 nM 31.25 nM 62.5 nM 125 nM PBS 5601 5601 5601 5601 146779 6491 6631 6027 5067 146786 5408 5328 4839 3518 505329 5719 5285 5384 4994 505330 7514 7262 6627 5179 505339 6572 6343 5349 4550 505347 7315 6602 6378 5908 505358 6357 6871 5798 5720 509927 5581 5487 5145 3601 509934 5476 5610 5394 4127 509958 5193 5492 5071 3957 509959 5051 5312 5144 3893 509960 4726 5160 5071 3305 509974 6913 7624 5798 5389 510038 5707 6381 5772 6733 510039 5981 7629 4802 6156 510040 4302 5209 5049 4188 510041 5565 5607 5205 3757 510100 8466 8378 7985 6402 510106 5703 5940 5231 4005 510116 5880 5380 4797 4757

Example 9 In vivo inhibition of HBV mRNA by MOE gapmers in HBV-transgenic mice

ISIS 146786, a 5-10-5 MOE gapmer, and ISIS 510100, a 3-10-4 MOE gapmer, both demonstrating significant inhibition of HBV mRNA, were tested in transgenic mice containing the HBV gene (Chisari 1.3.32 line) (Guidotti, L. G. et al., J. Virol. 1995, 69, 6158-6169) and the efficacy of the gapmers was evaluated.

Treatment

Two groups of ten-eleven HBV-transgenic male and female mice each were administered subcutaneously twice a week for four weeks with 25 mg/kg of ISIS 146786 or ISIS 510100. Another group of 14 male and female HBV-transgenic mice was administered Entecavir, an oral antiviral drug used to treat Hepatitis B infection, at 1 mg/kg daily for two weeks. Another group of 10 male and female HBV-transgenic female mice were injected subcutaneously with PBS twice a week for four weeks. The mice injected with PBS served as a control group. Liver HBV mRNA and DNA levels, plasma ALT, and body and organ weights were measured.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV using primer probe sets RTS3370, RTS3371, and RTS3372. Results are presented as percent inhibition of HBV mRNA, relative to PBS control. As shown in Table 20, treatment with ISIS antisense oligonucleotides resulted in significant reduction of HBV mRNA in comparison to the PBS control, irrespective of the primer probe set used for measurement. Entecavir did not decrease HBV mRNA expression.

TABLE 20 Inhibition of HBV mRNA in HBV-transgenic mice liver relative to the PBS control ISIS No RTS3370 RTS3371 RTS3372 146786 82 75 81 510100 93 83 89 DNA Analysis

DNA was extracted from liver tissue for real-time PCR analysis of HBV using primer probe sets RTS3370 and RTS3371. The levels were normalized to RIBOGREEN®. Results are presented as percent inhibition of HBV DNA, relative to PBS control. As shown in Table 21, treatment with ISIS antisense oligonucleotides resulted in significant reduction of HBV DNA in comparison to the PBS control, irrespective of the primer probe set used for measurement. Treatment with Entecavir also reduced DNA levels, as expected.

TABLE 21 Inhibition of HBV DNA in HBV-transgenic mice liver relative to the PBS control ISIS No RTS3370 RTS3371 146786 65 69 510100 67 73 Entecavir 75 96 Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminase was measured using a manual clinical chemistry analyzer (Teco Diagnostics, Anaheim, Calif.) Plasma concentrations of ALT (alanine transaminase) were measured and the results are presented in Table 22, expressed in IU/L. The results indicate that antisense inhibition of HBV had no adverse effects on the liver function of the mice.

TABLE 22 Effect of antisense oligonucleotide treatment on liver ALT of transgenic mice IU/mL PBS 12.7 ISIS 146786 24.1 ISIS 510100 25.8 Entecavir 23.7

The data from the study indicates that both ISIS 146786 and ISIS 510100 caused robust reductions in liver HBV RNA and DNA and treatment with these oligonucleotides were well tolerated in the transgenic mice.

Example 10 Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Several of the antisense oligonucleotides from the studies described above were also included in the assay. Cultured HepG2.2.15 cells at a density of 28,000 cells per well were transfected using LipofectAMINE 2000® reagent with 100 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in Table 23 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 23 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).

TABLE 23 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides targeted to SEQ ID NO: 1 (RTS3370) Viral Viral Start Stop % SEQ ID Site Site ISIS No Sequence inhibition NO 1 20 524410 TGGTGAAAGGTTGTGGAATT 70 321 4 23 524411 GTTTGGTGAAAGGTTGTGGA 51 322 7 26 524412 AGAGTTTGGTGAAAGGTTGT 47 323 10 29 524413 TGCAGAGTTTGGTGAAAGGT 74 324 13 32 524414 TCTTGCAGAGTTTGGTGAAA 91 325 16 35 524415 GGATCTTGCAGAGTTTGGTG 93 326 19 38 524416 CTGGGATCTTGCAGAGTTTG 85 327 22 41 524417 ACTCTGGGATCTTGCAGAGT 66 328 25 44 524418 CTCACTCTGGGATCTTGCAG 86 329 28 47 524419 CCTCTCACTCTGGGATCTTG 81 330 31 50 524420 AGGCCTCTCACTCTGGGATC 77 331 34 53 524421 TACAGGCCTCTCACTCTGGG 71 332 37 56 524422 AAATACAGGCCTCTCACTCT 68 333 40 59 524423 GGGAAATACAGGCCTCTCAC 43 334 43 62 524424 GCAGGGAAATACAGGCCTCT 76 335 46 65 524425 CCAGCAGGGAAATACAGGCC 89 336 49 68 524426 CCACCAGCAGGGAAATACAG 82 337 52 71 524427 GAGCCACCAGCAGGGAAATA 53 338 55 74 524428 CTGGAGCCACCAGCAGGGAA 76 339 56 75 524429 ACTGGAGCCACCAGCAGGGA 55 340 57 76 524430 AACTGGAGCCACCAGCAGGG 45 341 58 77 146779 GAACTGGAGCCACCAGCAGG 57 83 59 78 524431 TGAACTGGAGCCACCAGCAG 85 342 60 79 524432 CTGAACTGGAGCCACCAGCA 90 343 61 80 505314 CCTGAACTGGAGCCACCAGC 93 85 62 81 524433 TCCTGAACTGGAGCCACCAG 79 344 63 82 524434 CTCCTGAACTGGAGCCACCA 82 345 65 84 524435 TGCTCCTGAACTGGAGCCAC 78 346 68 87 524436 TACTGCTCCTGAACTGGAGC 58 347 71 90 524437 GTTTACTGCTCCTGAACTGG 40 348 74 93 524438 AGGGTTTACTGCTCCTGAAC 45 349 77 96 524439 AACAGGGTTTACTGCTCCTG 69 350 80 99 524440 CGGAACAGGGTTTACTGCTC 67 351 83 102 524441 AGTCGGAACAGGGTTTACTG 47 352 86 105 524442 AGTAGTCGGAACAGGGTTTA 59 353 89 108 524443 GGCAGTAGTCGGAACAGGGT 47 354 92 111 524444 AGAGGCAGTAGTCGGAACAG 54 355 95 114 524445 GGGAGAGGCAGTAGTCGGAA 49 356 98 117 524446 TAAGGGAGAGGCAGTAGTCG 81 357 101 120 524447 CGATAAGGGAGAGGCAGTAG 86 358 104 123 524448 TGACGATAAGGGAGAGGCAG 79 359 107 126 524449 GATTGACGATAAGGGAGAGG 27 360 110 129 524450 GAAGATTGACGATAAGGGAG 53 361 113 132 524451 CGAGAAGATTGACGATAAGG 67 362 116 135 524452 CCTCGAGAAGATTGACGATA 84 363 119 138 524453 AATCCTCGAGAAGATTGACG 79 364 122 141 524454 CCCAATCCTCGAGAAGATTG 65 365 125 144 524455 GTCCCCAATCCTCGAGAAGA 66 366 128 147 524456 AGGGTCCCCAATCCTCGAGA 67 367 131 150 524457 CGCAGGGTCCCCAATCCTCG 76 368 134 153 524458 CAGCGCAGGGTCCCCAATCC 59 369 137 156 524459 GTTCAGCGCAGGGTCCCCAA 80 370 140 159 524460 CATGTTCAGCGCAGGGTCCC 90 371 143 162 524461 CTCCATGTTCAGCGCAGGGT 75 372 146 165 524462 GTTCTCCATGTTCAGCGCAG 54 373 149 168 524463 GATGTTCTCCATGTTCAGCG 27 374 152 171 524464 TGTGATGTTCTCCATGTTCA 72 375 158 177 524466 TCCTGATGTGATGTTCTCCA 91 376 161 180 524467 GAATCCTGATGTGATGTTCT 77 377 164 183 524468 TAGGAATCCTGATGTGATGT 77 378 167 186 524469 TCCTAGGAATCCTGATGTGA 94 379 170 189 524470 GGGTCCTAGGAATCCTGATG 56 380 188 207 524471 CGCCTGTAACACGAGAAGGG 65 381 191 210 524472 CCCCGCCTGTAACACGAGAA 71 382 194 213 524473 AAACCCCGCCTGTAACACGA 74 383 195 214 524474 AAAACCCCGCCTGTAACACG 72 384 196 215 505315 AAAAACCCCGCCTGTAACAC 52 87 197 216 524475 GAAAAACCCCGCCTGTAACA 38 385 198 217 524476 AGAAAAACCCCGCCTGTAAC 18 386 200 219 524477 CAAGAAAAACCCCGCCTGTA 86 387 203 222 524478 CAACAAGAAAAACCCCGCCT 84 388 204 223 524479 TCAACAAGAAAAACCCCGCC 80 389 205 224 505317 GTCAACAAGAAAAACCCCGC 84 89 206 225 524480 TGTCAACAAGAAAAACCCCG 79 390 207 226 524481 TTGTCAACAAGAAAAACCCC 76 391 209 228 524482 TCTTGTCAACAAGAAAAACC 86 392 212 231 524483 GATTCTTGTCAACAAGAAAA 57 393 215 234 524484 GAGGATTCTTGTCAACAAGA 51 394 218 237 524485 TGTGAGGATTCTTGTCAACA 83 395 221 240 524486 TATTGTGAGGATTCTTGTCA 61 396 224 243 524487 CGGTATTGTGAGGATTCTTG 74 397 227 246 524488 CTGCGGTATTGTGAGGATTC 49 398 230 249 524489 ACTCTGCGGTATTGTGAGGA 67 399 233 252 524490 TAGACTCTGCGGTATTGTGA 88 400 236 255 524491 GTCTAGACTCTGCGGTATTG 84 401 239 258 524492 CGAGTCTAGACTCTGCGGTA 82 402 242 261 524493 CCACGAGTCTAGACTCTGCG 94 403 243 262 524494 ACCACGAGTCTAGACTCTGC 87 404 244 263 146821 CACCACGAGTCTAGACTCTG 87 92 245 264 524495 CCACCACGAGTCTAGACTCT 80 405 246 265 524496 TCCACCACGAGTCTAGACTC 65 406 247 266 505318 GTCCACCACGAGTCTAGACT 65 96 248 267 524497 AGTCCACCACGAGTCTAGAC 46 407 249 268 524498 AAGTCCACCACGAGTCTAGA 54 408 250 269 509921 GAAGTCCACCACGAGTCTAG 35 97 251 270 509922 AGAAGTCCACCACGAGTCTA 51 99 252 271 509923 GAGAAGTCCACCACGAGTCT 49 101 253 272 505319 AGAGAAGTCCACCACGAGTC 60 103 254 273 509924 GAGAGAAGTCCACCACGAGT 46 105 255 274 509925 TGAGAGAAGTCCACCACGAG 79 108 256 275 505320 TTGAGAGAAGTCCACCACGA 84 111 257 276 524499 ATTGAGAGAAGTCCACCACG 83 409 260 279 524500 AAAATTGAGAGAAGTCCACC 71 410 263 282 524501 TAGAAAATTGAGAGAAGTCC 67 411 266 285 524502 CCCTAGAAAATTGAGAGAAG 88 412 269 288 524503 TCCCCCTAGAAAATTGAGAG 82 413 272 291 524504 AGTTCCCCCTAGAAAATTGA 66 414 275 294 524505 GGTAGTTCCCCCTAGAAAAT 0 415 278 297 524506 CACGGTAGTTCCCCCTAGAA 65 416 281 300 524507 ACACACGGTAGTTCCCCCTA 87 417 284 303 524508 AAGACACACGGTAGTTCCCC 76 418 287 306 524509 GCCAAGACACACGGTAGTTC 61 419 290 309 524510 TTGGCCAAGACACACGGTAG 87 420 291 310 524511 TTTGGCCAAGACACACGGTA 87 421 292 311 524512 TTTTGGCCAAGACACACGGT 93 422 293 312 505323 ATTTTGGCCAAGACACACGG 83 123 294 313 524513 AATTTTGGCCAAGACACACG 79 423 295 314 524514 GAATTTTGGCCAAGACACAC 74 424 298 317 524515 TGCGAATTTTGGCCAAGACA 78 425 300 319 524516 ACTGCGAATTTTGGCCAAGA 71 426 301 320 524517 GACTGCGAATTTTGGCCAAG 71 427 302 321 505325 GGACTGCGAATTTTGGCCAA 50 125 303 322 524518 GGGACTGCGAATTTTGGCCA 55 428 321 340 524519 GTGAGTGATTGGAGGTTGGG 68 429 324 343 524520 TTGGTGAGTGATTGGAGGTT 84 430 327 346 524521 AGGTTGGTGAGTGATTGGAG 64 431 330 349 524522 AGGAGGTTGGTGAGTGATTG 58 432 333 352 524523 GACAGGAGGTTGGTGAGTGA 62 433 336 355 524524 GAGGACAGGAGGTTGGTGAG 56 434 339 358 524525 TTGGAGGACAGGAGGTTGGT 81 435 342 361 524526 AAGTTGGAGGACAGGAGGTT 77 436 345 364 524527 GACAAGTTGGAGGACAGGAG 69 437 348 367 524528 CAGGACAAGTTGGAGGACAG 82 438 351 370 524529 AACCAGGACAAGTTGGAGGA 67 439 354 373 524530 GATAACCAGGACAAGTTGGA 53 440 357 376 524531 AGCGATAACCAGGACAAGTT 55 441 358 377 524532 CAGCGATAACCAGGACAAGT 84 442 359 378 524533 CCAGCGATAACCAGGACAAG 86 443 360 379 505326 TCCAGCGATAACCAGGACAA 79 126 361 380 524534 ATCCAGCGATAACCAGGACA 85 444 362 381 524535 CATCCAGCGATAACCAGGAC 90 445 364 383 524536 CACATCCAGCGATAACCAGG 82 446 365 384 524537 ACACATCCAGCGATAACCAG 72 447 366 385 505327 GACACATCCAGCGATAACCA 61 127 367 386 524538 AGACACATCCAGCGATAACC 79 448 368 387 524539 CAGACACATCCAGCGATAAC 73 449 370 389 524540 CGCAGACACATCCAGCGATA 94 450 373 392 524541 CGCCGCAGACACATCCAGCG 84 451 390 409 524542 AGAGGAAGATGATAAAACGC 45 452 393 412 524543 TGAAGAGGAAGATGATAAAA 62 453 396 415 524544 GGATGAAGAGGAAGATGATA 58 454 399 418 524545 GCAGGATGAAGAGGAAGATG 48 455 402 421 524546 GCAGCAGGATGAAGAGGAAG 60 456 405 424 524547 ATAGCAGCAGGATGAAGAGG 84 457 408 427 524548 GGCATAGCAGCAGGATGAAG 56 458 409 428 524549 AGGCATAGCAGCAGGATGAA 78 459 410 429 524550 GAGGCATAGCAGCAGGATGA 67 460 411 430 505329 TGAGGCATAGCAGCAGGATG 85 136 412 431 509926 ATGAGGCATAGCAGCAGGAT 84 139 413 432 509927 GATGAGGCATAGCAGCAGGA 68 142 414 433 505330 AGATGAGGCATAGCAGCAGG 82 20 415 434 509928 AAGATGAGGCATAGCAGCAG 83 22 416 435 509929 GAAGATGAGGCATAGCAGCA 80 24 417 436 509930 AGAAGATGAGGCATAGCAGC 78 26 418 437 146783 AAGAAGATGAGGCATAGCAG 80 28 419 438 524551 CAAGAAGATGAGGCATAGCA 55 461 422 441 524552 CAACAAGAAGATGAGGCATA 90 462 425 444 524553 AACCAACAAGAAGATGAGGC 82 463 428 447 524554 AAGAACCAACAAGAAGATGA 79 464 431 450 524555 CAGAAGAACCAACAAGAAGA 72 465 434 453 524556 GTCCAGAAGAACCAACAAGA 87 466 437 456 524557 ATAGTCCAGAAGAACCAACA 72 467 440 459 524558 TTGATAGTCCAGAAGAACCA 76 468 443 462 524559 ACCTTGATAGTCCAGAAGAA 78 469 446 465 524560 CATACCTTGATAGTCCAGAA 77 470 449 468 524561 CAACATACCTTGATAGTCCA 69 471 452 471 524562 GGGCAACATACCTTGATAGT 39 472 455 474 524563 AACGGGCAACATACCTTGAT 72 473 456 475 524564 AAACGGGCAACATACCTTGA 86 474 457 476 505332 CAAACGGGCAACATACCTTG 85 166 458 477 524565 ACAAACGGGCAACATACCTT 80 475 459 478 524566 GACAAACGGGCAACATACCT 42 476 461 480 524567 AGGACAAACGGGCAACATAC 47 477 464 483 524568 TAGAGGACAAACGGGCAACA 81 478 467 486 524569 AATTAGAGGACAAACGGGCA 72 479 470 489 524570 TGGAATTAGAGGACAAACGG 84 480 471 490 524571 CTGGAATTAGAGGACAAACG 86 481 472 491 505335 CCTGGAATTAGAGGACAAAC 89 174 473 492 524572 TCCTGGAATTAGAGGACAAA 92 482 474 493 524573 ATCCTGGAATTAGAGGACAA 86 483 476 495 524574 GGATCCTGGAATTAGAGGAC 76 484 479 498 524575 TGAGGATCCTGGAATTAGAG 77 485 482 501 524576 GGTTGAGGATCCTGGAATTA 62 486 485 504 524577 GGTGGTTGAGGATCCTGGAA 73 487 488 507 524578 GCTGGTGGTTGAGGATCCTG 84 488 491 510 524579 CGTGCTGGTGGTTGAGGATC 79 489 494 513 524580 TCCCGTGCTGGTGGTTGAGG 83 490 497 516 524581 TGGTCCCGTGCTGGTGGTTG 66 491 500 519 524582 GCATGGTCCCGTGCTGGTGG 77 492 503 522 524583 TCGGCATGGTCCCGTGCTGG 0 493 506 525 524584 GGTTCGGCATGGTCCCGTGC 56 494 509 528 524585 GCAGGTTCGGCATGGTCCCG 61 495 512 531 524586 CATGCAGGTTCGGCATGGTC 87 496 515 534 524587 AGTCATGCAGGTTCGGCATG 77 497 518 537 524588 AGTAGTCATGCAGGTTCGGC 64 498 521 540 524589 AGCAGTAGTCATGCAGGTTC 61 499 524 543 524590 TTGAGCAGTAGTCATGCAGG 86 500 527 546 524591 TCCTTGAGCAGTAGTCATGC 80 501 530 549 524592 GGTTCCTTGAGCAGTAGTCA 50 502 533 552 524593 AGAGGTTCCTTGAGCAGTAG 61 503 536 555 524594 CATAGAGGTTCCTTGAGCAG 89 504 539 558 524595 ATACATAGAGGTTCCTTGAG 87 505 542 561 524596 GGGATACATAGAGGTTCCTT 0 506 545 564 524597 GGAGGGATACATAGAGGTTC 38 507 548 567 524598 ACAGGAGGGATACATAGAGG 73 508 551 570 524599 GCAACAGGAGGGATACATAG 67 509 554 573 524600 ACAGCAACAGGAGGGATACA 72 510 557 576 524601 GGTACAGCAACAGGAGGGAT 59 511 560 579 524602 TTTGGTACAGCAACAGGAGG 81 512 563 582 524603 AGGTTTGGTACAGCAACAGG 74 513 566 585 524604 CGAAGGTTTGGTACAGCAAC 85 514 569 588 524605 GTCCGAAGGTTTGGTACAGC 76 515 572 591 524606 TCCGTCCGAAGGTTTGGTAC 80 516 575 594 524607 ATTTCCGTCCGAAGGTTTGG 88 517 578 597 524608 GCAATTTCCGTCCGAAGGTT 50 518 581 600 524609 GGTGCAATTTCCGTCCGAAG 55 519 584 603 524610 ACAGGTGCAATTTCCGTCCG 81 520 587 606 524611 AATACAGGTGCAATTTCCGT 88 521 590 609 524612 GGGAATACAGGTGCAATTTC 32 522 593 612 524613 GATGGGAATACAGGTGCAAT 49 523 608 627 524614 AGCCCAGGATGATGGGATGG 89 524 611 630 524615 GAAAGCCCAGGATGATGGGA 71 525 614 633 524616 TCCGAAAGCCCAGGATGATG 86 526 617 636 524617 TTTTCCGAAAGCCCAGGATG 97 527 620 639 524618 GAATTTTCCGAAAGCCCAGG 80 528 623 642 524619 TAGGAATTTTCCGAAAGCCC 95 529 626 645 524620 CCATAGGAATTTTCCGAAAG 88 530 629 648 524621 CTCCCATAGGAATTTTCCGA 83 531 632 651 524622 CCACTCCCATAGGAATTTTC 68 532 635 654 524623 GGCCCACTCCCATAGGAATT 60 533 638 657 524624 TGAGGCCCACTCCCATAGGA 57 534 641 660 524625 GGCTGAGGCCCACTCCCATA 62 535 644 663 524626 ACGGGCTGAGGCCCACTCCC 57 536 647 666 524627 GAAACGGGCTGAGGCCCACT 62 537 650 669 524628 GGAGAAACGGGCTGAGGCCC 31 538 653 672 524629 CCAGGAGAAACGGGCTGAGG 77 539 656 675 524630 GAGCCAGGAGAAACGGGCTG 48 540 659 678 524631 ACTGAGCCAGGAGAAACGGG 43 541 662 681 524632 TAAACTGAGCCAGGAGAAAC 67 542 665 684 524633 TAGTAAACTGAGCCAGGAGA 86 543 668 687 524634 CACTAGTAAACTGAGCCAGG 96 544 669 688 524635 GCACTAGTAAACTGAGCCAG 83 545 671 690 524636 TGGCACTAGTAAACTGAGCC 84 546 672 691 524637 ATGGCACTAGTAAACTGAGC 82 547 674 693 524638 AAATGGCACTAGTAAACTGA 74 548 677 696 524639 AACAAATGGCACTAGTAAAC 63 549 678 697 524640 GAACAAATGGCACTAGTAAA 67 550 679 698 505338 TGAACAAATGGCACTAGTAA 84 186 680 699 524641 CTGAACAAATGGCACTAGTA 95 551 681 700 524642 ACTGAACAAATGGCACTAGT 77 552 682 701 505339 CACTGAACAAATGGCACTAG 95 187 683 702 524643 CCACTGAACAAATGGCACTA 89 553 684 703 524644 ACCACTGAACAAATGGCACT 90 554 686 705 524646 GAACCACTGAACAAATGGCA 82 555 687 706 509931 CGAACCACTGAACAAATGGC 90 39 689 708 524647 TACGAACCACTGAACAAATG 79 556 690 709 146824 CTACGAACCACTGAACAAAT 72 557 692 711 524648 CCCTACGAACCACTGAACAA 73 558 693 712 524649 GCCCTACGAACCACTGAACA 83 559 695 714 524650 AAGCCCTACGAACCACTGAA 82 560 696 715 524651 AAAGCCCTACGAACCACTGA 81 561 697 716 505342 GAAAGCCCTACGAACCACTG 66 198 698 717 524652 GGAAAGCCCTACGAACCACT 59 562 699 718 524653 GGGAAAGCCCTACGAACCAC 46 563 718 737 524654 ACTGAAAGCCAAACAGTGGG 64 564 721 740 524655 ATAACTGAAAGCCAAACAGT 0 565 724 743 524656 CATATAACTGAAAGCCAAAC 70 566 727 746 524657 ATCCATATAACTGAAAGCCA 91 567 730 749 524658 ATCATCCATATAACTGAAAG 69 568 733 752 524659 CACATCATCCATATAACTGA 70 569 736 755 524660 TACCACATCATCCATATAAC 57 570 739 758 524661 CAATACCACATCATCCATAT 70 571 742 761 524662 CCCCAATACCACATCATCCA 85 572 745 764 524663 GGCCCCCAATACCACATCAT 70 573 748 767 524664 CTTGGCCCCCAATACCACAT 82 574 751 770 524665 AGACTTGGCCCCCAATACCA 77 575 754 773 524666 TACAGACTTGGCCCCCAATA 77 576 757 776 524667 CTGTACAGACTTGGCCCCCA 90 577 760 779 524668 ATGCTGTACAGACTTGGCCC 79 578 763 782 524669 AAGATGCTGTACAGACTTGG 79 579 766 785 524670 CTCAAGATGCTGTACAGACT 84 580 769 788 524671 GGACTCAAGATGCTGTACAG 24 581 772 791 524672 AAGGGACTCAAGATGCTGTA 57 582 775 794 524673 AAAAAGGGACTCAAGATGCT 66 583 778 797 524674 GGTAAAAAGGGACTCAAGAT 30 584 781 800 524675 AGCGGTAAAAAGGGACTCAA 68 585 784 803 524676 AACAGCGGTAAAAAGGGACT 67 586 787 806 524677 GGTAACAGCGGTAAAAAGGG 48 587 790 809 524678 ATTGGTAACAGCGGTAAAAA 81 588 793 812 524679 AAAATTGGTAACAGCGGTAA 89 589 796 815 524680 AAGAAAATTGGTAACAGCGG 84 590 799 818 524681 CAAAAGAAAATTGGTAACAG 41 591 802 821 524682 AGACAAAAGAAAATTGGTAA 51 592 805 824 524683 CAAAGACAAAAGAAAATTGG 66 593 808 827 524684 ACCCAAAGACAAAAGAAAAT 61 594 811 830 524685 TATACCCAAAGACAAAAGAA 79 595 814 833 524686 ATGTATACCCAAAGACAAAA 84 596 817 836 524687 TAAATGTATACCCAAAGACA 77 597 820 839 524688 GTTTAAATGTATACCCAAAG 80 598 821 840 524689 GGTTTAAATGTATACCCAAA 71 599 822 841 524690 GGGTTTAAATGTATACCCAA 85 600 823 842 505344 AGGGTTTAAATGTATACCCA 85 206 824 843 524691 TAGGGTTTAAATGTATACCC 90 601 825 844 524692 TTAGGGTTTAAATGTATACC 83 602 827 846 524693 TGTTAGGGTTTAAATGTATA 53 603 830 849 524694 TTTTGTTAGGGTTTAAATGT 67 604 845 864 524695 AACCCCATCTCTTTGTTTTG 81 605 848 867 524696 AGTAACCCCATCTCTTTGTT 71 606 851 870 524697 GAGAGTAACCCCATCTCTTT 65 607 854 873 524698 TCAGAGAGTAACCCCATCTC 96 608 857 876 524699 AATTCAGAGAGTAACCCCAT 94 609 860 879 524700 TAAAATTCAGAGAGTAACCC 71 610 863 882 524701 CCATAAAATTCAGAGAGTAA 90 611 866 885 524702 AACCCATAAAATTCAGAGAG 86 612 869 888 524703 CATAACCCATAAAATTCAGA 72 613 872 891 524704 TGACATAACCCATAAAATTC 81 614 875 894 524705 CAATGACATAACCCATAAAA 81 615 878 897 524706 TTCCAATGACATAACCCATA 95 616 881 900 524707 AACTTCCAATGACATAACCC 91 617 884 903 524708 CATAACTTCCAATGACATAA 83 618 887 906 524709 ACCCATAACTTCCAATGACA 95 619 890 909 524710 AGGACCCATAACTTCCAATG 66 620 893 912 524711 GCAAGGACCCATAACTTCCA 41 621 896 915 524712 GTGGCAAGGACCCATAACTT 53 622 899 918 524713 CTTGTGGCAAGGACCCATAA 91 623 902 921 524714 GTTCTTGTGGCAAGGACCCA 77 624 905 924 524715 TGTGTTCTTGTGGCAAGGAC 90 625 908 927 524716 TGATGTGTTCTTGTGGCAAG 90 626 911 930 524717 GTATGATGTGTTCTTGTGGC 82 627 914 933 524718 TTTGTATGATGTGTTCTTGT 95 628 930 949 524719 AAACATTCTTTGATTTTTTG 61 629 933 952 524720 CTAAAACATTCTTTGATTTT 43 630 936 955 524721 TTTCTAAAACATTCTTTGAT 90 631 939 958 524722 AGTTTTCTAAAACATTCTTT 75 632 942 961 524723 GGAAGTTTTCTAAAACATTC 52 633 945 964 524724 ATAGGAAGTTTTCTAAAACA 74 634 948 967 524725 TTAATAGGAAGTTTTCTAAA 40 635 951 970 524726 CTGTTAATAGGAAGTTTTCT 93 636 954 973 524727 GGCCTGTTAATAGGAAGTTT 87 637 957 976 524728 ATAGGCCTGTTAATAGGAAG 85 638 960 979 524729 TCAATAGGCCTGTTAATAGG 92 639 963 982 524730 CAATCAATAGGCCTGTTAAT 90 640 966 985 524731 TTCCAATCAATAGGCCTGTT 96 641 969 988 524732 ACTTTCCAATCAATAGGCCT 77 642 972 991 146826 CATACTTTCCAATCAATAGG 92 643 975 994 524733 TGACATACTTTCCAATCAAT 91 644 978 997 524734 CGTTGACATACTTTCCAATC 95 645 996 1015 524735 CCCAAAAGACCCACAATTCG 92 646 999 1018 524736 AAACCCAAAAGACCCACAAT 74 647 1002 1021 524737 GCAAAACCCAAAAGACCCAC 85 648 1005 1024 524738 GCAGCAAAACCCAAAAGACC 70 649 1025 1044 524739 AACCACATTGTGTAAATGGG 90 650 1028 1047 524740 GATAACCACATTGTGTAAAT 58 651 1031 1050 524741 CAGGATAACCACATTGTGTA 83 652 1034 1053 524742 ACGCAGGATAACCACATTGT 84 653 1037 1056 524743 TTAACGCAGGATAACCACAT 93 654 1040 1059 524744 GCATTAACGCAGGATAACCA 60 655 1043 1062 524745 AGGGCATTAACGCAGGATAA 58 656 1046 1065 524746 ACAAGGGCATTAACGCAGGA 75 657 1049 1068 524747 CATACAAGGGCATTAACGCA 89 658 1052 1071 524748 ATGCATACAAGGGCATTAAC 87 659 1055 1074 524749 TACATGCATACAAGGGCATT 86 660 1058 1077 524750 GAATACATGCATACAAGGGC 75 661 1061 1080 524751 ATTGAATACATGCATACAAG 81 662 1064 1083 524752 TAGATTGAATACATGCATAC 85 663 1067 1086 524753 GCTTAGATTGAATACATGCA 69 664 1070 1089 524754 CCTGCTTAGATTGAATACAT 90 665 1073 1092 524755 AAGCCTGCTTAGATTGAATA 76 666 1076 1095 524756 TGAAAGCCTGCTTAGATTGA 76 667 1079 1098 524757 AAGTGAAAGCCTGCTTAGAT 68 668 1082 1101 524758 AGAAAGTGAAAGCCTGCTTA 81 669 1085 1104 524759 GCGAGAAAGTGAAAGCCTGC 61 670 1088 1107 524760 TTGGCGAGAAAGTGAAAGCC 89 671 1091 1110 524761 AAGTTGGCGAGAAAGTGAAA 74 672 1094 1113 524762 TGTAAGTTGGCGAGAAAGTG 85 673 1097 1116 524763 CCTTGTAAGTTGGCGAGAAA 90 674 1100 1119 524764 AGGCCTTGTAAGTTGGCGAG 93 675 1103 1122 524765 GAAAGGCCTTGTAAGTTGGC 78 676 1106 1125 524766 ACAGAAAGGCCTTGTAAGTT 76 677 1109 1128 524767 TACACAGAAAGGCCTTGTAA 94 678 1112 1131 524768 GTTTACACAGAAAGGCCTTG 80 679 1115 1134 524769 ATTGTTTACACAGAAAGGCC 83 680 1118 1137 524770 GGTATTGTTTACACAGAAAG 63 681 1121 1140 524771 TCAGGTATTGTTTACACAGA 93 682 1124 1143 524772 GGTTCAGGTATTGTTTACAC 68 683 1127 1146 524773 AAAGGTTCAGGTATTGTTTA 82 684 1130 1149 524774 GGTAAAGGTTCAGGTATTGT 68 685 1150 1169 524775 TGGCCGTTGCCGGGCAACGG 74 686 1153 1172 524776 ACCTGGCCGTTGCCGGGCAA 77 687 1156 1175 524777 CAGACCTGGCCGTTGCCGGG 88 688 1159 1178 524778 GCACAGACCTGGCCGTTGCC 80 689 1162 1181 524779 TTGGCACAGACCTGGCCGTT 85 690 1165 1184 524780 CACTTGGCACAGACCTGGCC 93 691 1168 1187 524781 AAACACTTGGCACAGACCTG 90 692 1169 1188 524782 CAAACACTTGGCACAGACCT 75 693 1170 1189 505345 GCAAACACTTGGCACAGACC 78 207 1171 1190 524783 AGCAAACACTTGGCACAGAC 84 694 1172 1191 524784 CAGCAAACACTTGGCACAGA 90 695 1174 1193 524785 GTCAGCAAACACTTGGCACA 79 696 1200 1219 524786 ACCAAGCCCCAGCCAGTGGG 57 697 1203 1222 524787 ATGACCAAGCCCCAGCCAGT 74 698 1206 1225 524788 CCCATGACCAAGCCCCAGCC 90 699 1209 1228 524789 TGGCCCATGACCAAGCCCCA 96 700 1212 1231 524790 TGATGGCCCATGACCAAGCC 79 701 1215 1234 524791 CGCTGATGGCCCATGACCAA 97 702 1218 1237 524792 ACGCGCTGATGGCCCATGAC 98 703 1221 1240 524793 CGCACGCGCTGATGGCCCAT 98 704 1224 1243 524794 CCACGCACGCGCTGATGGCC 98 705 1227 1246 524795 GTTCCACGCACGCGCTGATG 98 706 1230 1249 524796 AAGGTTCCACGCACGCGCTG 99 707 1233 1252 524797 GAAAAGGTTCCACGCACGCG 97 708 1236 1255 524798 GCCGAAAAGGTTCCACGCAC 98 709 1239 1258 524799 GGAGCCGAAAAGGTTCCACG 75 710 1242 1261 524800 AGAGGAGCCGAAAAGGTTCC 79 711 1245 1264 524801 GGCAGAGGAGCCGAAAAGGT 98 712 1248 1267 524802 ATCGGCAGAGGAGCCGAAAA 73 713 1251 1270 524803 TGGATCGGCAGAGGAGCCGA 91 714 1254 1273 524804 GTATGGATCGGCAGAGGAGC 98 715 1257 1276 524805 GCAGTATGGATCGGCAGAGG 98 716 1258 1277 524806 CGCAGTATGGATCGGCAGAG 98 717 1259 1278 505346 CCGCAGTATGGATCGGCAGA 98 210 1260 1279 146785 TCCGCAGTATGGATCGGCAG 98 718 1261 1280 524807 TTCCGCAGTATGGATCGGCA 98 719 1262 1281 505347 GTTCCGCAGTATGGATCGGC 98 212 1263 1282 524808 AGTTCCGCAGTATGGATCGG 96 720 1264 1283 524809 GAGTTCCGCAGTATGGATCG 97 721 1266 1285 524810 AGGAGTTCCGCAGTATGGAT 96 722 1269 1288 524811 GCTAGGAGTTCCGCAGTATG 96 723 1272 1291 524812 GCGGCTAGGAGTTCCGCAGT 75 724 1275 1294 524813 CAAGCGGCTAGGAGTTCCGC 86 725 1278 1297 524814 AAACAAGCGGCTAGGAGTTC 73 726 1281 1300 524815 GCAAAACAAGCGGCTAGGAG 71 727 1282 1301 524816 AGCAAAACAAGCGGCTAGGA 89 728 1283 1302 505352 GAGCAAAACAAGCGGCTAGG 76 217 1284 1303 524817 CGAGCAAAACAAGCGGCTAG 78 729 1285 1304 524818 GCGAGCAAAACAAGCGGCTA 71 730 1286 1305 505353 TGCGAGCAAAACAAGCGGCT 82 218 1287 1306 524819 CTGCGAGCAAAACAAGCGGC 82 731 1288 1307 524820 GCTGCGAGCAAAACAAGCGG 67 732 1290 1309 524821 CTGCTGCGAGCAAAACAAGC 79 733 1293 1312 524822 GACCTGCTGCGAGCAAAACA 87 734 1296 1315 524823 CCAGACCTGCTGCGAGCAAA 94 735 1299 1318 524824 GCTCCAGACCTGCTGCGAGC 80 736 1302 1321 524825 TTTGCTCCAGACCTGCTGCG 70 737 1305 1324 524826 ATGTTTGCTCCAGACCTGCT 75 738 1308 1327 524827 ATAATGTTTGCTCCAGACCT 55 739 1311 1330 524828 CCGATAATGTTTGCTCCAGA 87 740 1314 1333 524829 GTCCCGATAATGTTTGCTCC 80 741 1317 1336 524830 TCAGTCCCGATAATGTTTGC 76 742 1320 1339 524831 TTATCAGTCCCGATAATGTT 53 743 1577 1596 146786 GTGAAGCGAAGTGCACACGG 96 224

Example 11 Antisense Inhibition of HBV Viral mRNA in HepG2.2.15 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. Several of the antisense oligonucleotides from the studies described above were also included in the assay. Cultured HepG2.2.15 cells at a density of 28,000 cells per well were transfected using LipofectAMINE 2000® reagent with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. The mRNA levels of some of the gapmers were also measured using RTS3372. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in Tables 24 and 25 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. Each nucleoside in the central gap segment has a deoxy sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 24 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). Each gapmer listed in Table 25 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1286 (a permuted version of GENBANK Accession No. U95551.1). ‘n/a’ indicates that the inhibition data for that particular gapmer was not measured with that particular primer probe set.

TABLE 24 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides targeted to SEQ ID NO: 1 (RTS3370 and RTS3372) SEQ Start Stop RTS3370 % RTS3372 % ID Site Site ISIS No Sequence inhibition inhibition NO 1323 1342 524832 GAGTTATCAGTCCCGATAAT 63 n/a 744 1326 1345 524833 ACAGAGTTATCAGTCCCGAT 82 n/a 745 1329 1348 524834 ACAACAGAGTTATCAGTCCC 52 n/a 746 1332 1351 524835 AGGACAACAGAGTTATCAGT 57 n/a 747 1335 1354 524836 GAGAGGACAACAGAGTTATC 49 n/a 748 1338 1357 524837 CGGGAGAGGACAACAGAGTT 0 n/a 749 1341 1360 524838 TTGCGGGAGAGGACAACAGA 17 n/a 750 1344 1363 524839 TATTTGCGGGAGAGGACAAC 30 n/a 751 1347 1366 524840 GTATATTTGCGGGAGAGGAC 22 n/a 752 1350 1369 524841 GATGTATATTTGCGGGAGAG 32 n/a 753 1353 1372 524842 TACGATGTATATTTGCGGGA 76 n/a 754 1356 1375 524843 GGATACGATGTATATTTGCG 76 n/a 755 1359 1378 524844 CATGGATACGATGTATATTT 87 n/a 756 1362 1381 524845 AGCCATGGATACGATGTATA 70 n/a 757 1365 1384 524846 AGCAGCCATGGATACGATGT 22 n/a 758 1368 1387 524847 CCTAGCAGCCATGGATACGA 67 n/a 759 1371 1390 524848 CAGCCTAGCAGCCATGGATA 56 n/a 760 1374 1393 524849 GCACAGCCTAGCAGCCATGG 38 n/a 761 1377 1396 524850 GCAGCACAGCCTAGCAGCCA 11 n/a 762 1380 1399 524851 TTGGCAGCACAGCCTAGCAG 34 n/a 763 1383 1402 524852 CAGTTGGCAGCACAGCCTAG 47 n/a 764 1386 1405 524853 ATCCAGTTGGCAGCACAGCC 45 n/a 765 1389 1408 524854 AGGATCCAGTTGGCAGCACA 36 n/a 766 1392 1411 524855 CGCAGGATCCAGTTGGCAGC 41 n/a 767 1395 1414 524856 CCGCGCAGGATCCAGTTGGC 72 n/a 768 1398 1417 524857 GTCCCGCGCAGGATCCAGTT 55 n/a 769 1457 1476 524858 AGCGACCCCGAGAAGGGTCG 17 n/a 770 1460 1479 524859 CCAAGCGACCCCGAGAAGGG 45 n/a 771 1463 1482 524860 GTCCCAAGCGACCCCGAGAA 8 n/a 772 1466 1485 524861 AGAGTCCCAAGCGACCCCGA 51 n/a 773 1469 1488 524862 GAGAGAGTCCCAAGCGACCC 28 n/a 774 1472 1491 524863 GACGAGAGAGTCCCAAGCGA 37 n/a 775 1492 1511 524864 GAACGGCAGACGGAGAAGGG 27 n/a 776 1498 1517 524866 CGGTCGGAACGGCAGACGGA 78 n/a 777 1501 1520 524867 GGTCGGTCGGAACGGCAGAC 78 n/a 778 1504 1523 524868 CGTGGTCGGTCGGAACGGCA 79 n/a 779 1507 1526 524869 CCCCGTGGTCGGTCGGAACG 70 n/a 780 1510 1529 524870 GCGCCCCGTGGTCGGTCGGA 78 n/a 781 1513 1532 524871 GGTGCGCCCCGTGGTCGGTC 74 n/a 782 1514 1533 524872 AGGTGCGCCCCGTGGTCGGT 63 n/a 783 1515 1534 505354 GAGGTGCGCCCCGTGGTCGG 70 n/a 220 1516 1535 524873 AGAGGTGCGCCCCGTGGTCG 72 n/a 784 1517 1536 524874 GAGAGGTGCGCCCCGTGGTC 49 n/a 785 1518 1537 505355 AGAGAGGTGCGCCCCGTGGT 64 n/a 221 1519 1538 524875 AAGAGAGGTGCGCCCCGTGG 57 n/a 786 1520 1539 524876 AAAGAGAGGTGCGCCCCGTG 63 n/a 787 1521 1540 505356 TAAAGAGAGGTGCGCCCCGT 68 n/a 222 1522 1541 524877 GTAAAGAGAGGTGCGCCCCG 50 n/a 788 1523 1542 524878 CGTAAAGAGAGGTGCGCCCC 64 n/a 789 1550 1569 524879 GATGAGAAGGCACAGACGGG 70 n/a 790 1553 1572 524880 GCAGATGAGAAGGCACAGAC 81 n/a 791 1556 1575 524881 CCGGCAGATGAGAAGGCACA 80 n/a 792 1559 1578 524882 GGTCCGGCAGATGAGAAGGC 84 n/a 793 1562 1581 524883 CACGGTCCGGCAGATGAGAA 79 n/a 794 1565 1584 524884 GCACACGGTCCGGCAGATGA 83 n/a 795 1568 1587 524885 AGTGCACACGGTCCGGCAGA 77 n/a 796 1571 1590 524886 CGAAGTGCACACGGTCCGGC 89 n/a 797 1574 1593 524887 AAGCGAAGTGCACACGGTCC 85 n/a 798 1575 1594 524888 GAAGCGAAGTGCACACGGTC 83 n/a 799 1576 1595 524889 TGAAGCGAAGTGCACACGGT 83 n/a 800 1577 1596 146786 GTGAAGCGAAGTGCACACGG 88 85 224 1578 1597 524890 GGTGAAGCGAAGTGCACACG 83 n/a 801 1579 1598 524891 AGGTGAAGCGAAGTGCACAC 82 n/a 802 1580 1599 505357 GAGGTGAAGCGAAGTGCACA 79 n/a 803 1581 1600 524892 AGAGGTGAAGCGAAGTGCAC 73 n/a 804 1582 1601 524893 CAGAGGTGAAGCGAAGTGCA 80 n/a 805 1583 1602 505358 GCAGAGGTGAAGCGAAGTGC 84 n/a 226 1584 1603 524894 TGCAGAGGTGAAGCGAAGTG 74 n/a 806 1585 1604 524895 GTGCAGAGGTGAAGCGAAGT 72 n/a 807 1586 1605 505359 CGTGCAGAGGTGAAGCGAAG 78 n/a 227 1604 1623 524896 ACGGTGGTCTCCATGCGACG 79 n/a 808 1607 1626 524897 TTCACGGTGGTCTCCATGCG 75 n/a 809 1630 1649 524898 CCTTGGGCAACATTCGGTGG 77 n/a 810 1633 1652 524899 AGACCTTGGGCAACATTCGG 76 n/a 811 1636 1655 524900 GTAAGACCTTGGGCAACATT 73 n/a 812 1639 1658 524901 TATGTAAGACCTTGGGCAAC 60 n/a 813 1642 1661 524902 TCTTATGTAAGACCTTGGGC 72 n/a 814 1645 1664 524903 TCCTCTTATGTAAGACCTTG 75 n/a 815 1648 1667 524904 GAGTCCTCTTATGTAAGACC 65 n/a 816 1651 1670 524905 CAAGAGTCCTCTTATGTAAG 76 n/a 817 1654 1673 524906 GTCCAAGAGTCCTCTTATGT 78 n/a 818 1657 1676 524907 AGAGTCCAAGAGTCCTCTTA 82 n/a 819 1660 1679 524908 CAGAGAGTCCAAGAGTCCTC 82 n/a 820 1663 1682 524909 TTGCAGAGAGTCCAAGAGTC 76 n/a 821 1666 1685 524910 ACATTGCAGAGAGTCCAAGA 76 n/a 822 1669 1688 524911 TTGACATTGCAGAGAGTCCA 74 n/a 823 1689 1708 524912 GTATGCCTCAAGGTCGGTCG 76 n/a 824 1692 1711 524913 GAAGTATGCCTCAAGGTCGG 73 n/a 825 1695 1714 524914 TTTGAAGTATGCCTCAAGGT 76 n/a 826 1698 1717 524915 GTCTTTGAAGTATGCCTCAA 75 n/a 827 1701 1720 524916 ACAGTCTTTGAAGTATGCCT 77 n/a 828 1704 1723 524917 CAAACAGTCTTTGAAGTATG 55 n/a 829 1707 1726 524918 AAACAAACAGTCTTTGAAGT 59 n/a 830 1710 1729 524919 TTTAAACAAACAGTCTTTGA 53 n/a 831 1713 1732 524920 GTCTTTAAACAAACAGTCTT 3 n/a 832 1716 1735 524921 CCAGTCTTTAAACAAACAGT 75 n/a 833 1719 1738 524922 CTCCCAGTCTTTAAACAAAC 70 n/a 834 1722 1741 524923 CTCCTCCCAGTCTTTAAACA 68 n/a 835 1725 1744 524924 CAACTCCTCCCAGTCTTTAA 62 n/a 836 1728 1747 524925 CCCCAACTCCTCCCAGTCTT 63 n/a 837 1731 1750 524926 CTCCCCCAACTCCTCCCAGT 62 n/a 838 1734 1753 524927 CTCCTCCCCCAACTCCTCCC 55 n/a 839 1737 1756 524928 AATCTCCTCCCCCAACTCCT 61 n/a 840 1740 1759 524929 TCTAATCTCCTCCCCCAACT 61 n/a 841 1743 1762 524930 TAATCTAATCTCCTCCCCCA 70 n/a 842 1746 1765 524931 CTTTAATCTAATCTCCTCCC 74 n/a 843 1749 1768 524932 GACCTTTAATCTAATCTCCT 74 n/a 844 1752 1771 524933 AAAGACCTTTAATCTAATCT 60 n/a 845 1755 1774 524934 TACAAAGACCTTTAATCTAA 55 n/a 846 1758 1777 524935 TAGTACAAAGACCTTTAATC 54 n/a 847 1761 1780 524936 TCCTAGTACAAAGACCTTTA 69 n/a 848 1764 1783 524937 GCCTCCTAGTACAAAGACCT 72 n/a 849 1767 1786 524938 ACAGCCTCCTAGTACAAAGA 60 n/a 850 1770 1789 524939 CCTACAGCCTCCTAGTACAA 66 n/a 851 1773 1792 524940 ATGCCTACAGCCTCCTAGTA 70 n/a 852 1776 1795 524941 TTTATGCCTACAGCCTCCTA 63 n/a 853 1777 1796 524942 ATTTATGCCTACAGCCTCCT 70 n/a 854 1778 1797 509932 AATTTATGCCTACAGCCTCC 68 n/a 46 1779 1798 509933 CAATTTATGCCTACAGCCTC 68 n/a 48 1780 1799 509934 CCAATTTATGCCTACAGCCT 65 n/a 50 1781 1800 509935 ACCAATTTATGCCTACAGCC 64 n/a 52 1782 1801 524943 GACCAATTTATGCCTACAGC 57 n/a 855 1783 1802 524944 AGACCAATTTATGCCTACAG 60 n/a 856 1785 1804 524945 GCAGACCAATTTATGCCTAC 54 n/a 857 1788 1807 524946 TGCGCAGACCAATTTATGCC 68 n/a 858 1791 1810 524947 TGGTGCGCAGACCAATTTAT 64 n/a 859 1794 1813 524948 TGCTGGTGCGCAGACCAATT 75 n/a 860 1797 1816 524949 TGGTGCTGGTGCGCAGACCA 68 n/a 861 1800 1819 524950 GCATGGTGCTGGTGCGCAGA 69 n/a 862 1803 1822 524951 GTTGCATGGTGCTGGTGCGC 59 n/a 863 1807 1826 524952 AAAAGTTGCATGGTGCTGGT 61 n/a 864 1810 1829 524953 TGAAAAAGTTGCATGGTGCT 60 n/a 865 1813 1832 524954 AGGTGAAAAAGTTGCATGGT 61 n/a 866 1816 1835 524955 CAGAGGTGAAAAAGTTGCAT 63 n/a 867 1819 1838 524956 AGGCAGAGGTGAAAAAGTTG 57 n/a 868 1822 1841 524957 ATTAGGCAGAGGTGAAAAAG 50 n/a 869 1823 1842 524958 GATTAGGCAGAGGTGAAAAA 57 n/a 870 1825 1844 524959 ATGATTAGGCAGAGGTGAAA 54 n/a 871 1828 1847 524960 GAGATGATTAGGCAGAGGTG 59 n/a 872 1831 1850 524961 CAAGAGATGATTAGGCAGAG 61 n/a 873 1834 1853 524962 GAACAAGAGATGATTAGGCA 56 n/a 874 1837 1856 524963 CATGAACAAGAGATGATTAG 24 n/a 875 1840 1859 524964 GGACATGAACAAGAGATGAT 54 n/a 876 1843 1862 524965 GTAGGACATGAACAAGAGAT 52 n/a 877 1846 1865 524966 ACAGTAGGACATGAACAAGA 47 n/a 878 1849 1868 524967 TGAACAGTAGGACATGAACA 33 n/a 879 1852 1871 524968 GCTTGAACAGTAGGACATGA 44 n/a 880 1855 1874 524969 GAGGCTTGAACAGTAGGACA 43 n/a 881 1858 1877 524970 TTGGAGGCTTGAACAGTAGG 28 n/a 882 1862 1881 524971 CAGCTTGGAGGCTTGAACAG 30 n/a 883 1871 1890 524972 CCCAAGGCACAGCTTGGAGG 38 n/a 884 1874 1893 524973 CCACCCAAGGCACAGCTTGG 47 n/a 885 1877 1896 524974 AAGCCACCCAAGGCACAGCT 49 n/a 886 1880 1899 524975 CCAAAGCCACCCAAGGCACA 32 n/a 887 1883 1902 524976 GCCCCAAAGCCACCCAAGGC 56 n/a 888 1886 1905 524977 CATGCCCCAAAGCCACCCAA 63 n/a 889 1889 1908 524978 GTCCATGCCCCAAAGCCACC 64 n/a 890 1892 1911 524979 GATGTCCATGCCCCAAAGCC 65 n/a 891 1895 1914 524980 GTCGATGTCCATGCCCCAAA 80 n/a 892 1898 1917 524981 AGGGTCGATGTCCATGCCCC 79 n/a 893 1901 1920 524982 ATAAGGGTCGATGTCCATGC 79 n/a 894 1904 1923 524983 TTTATAAGGGTCGATGTCCA 71 n/a 895 1907 1926 524984 TTCTTTATAAGGGTCGATGT 77 n/a 896 1910 1929 524985 AAATTCTTTATAAGGGTCGA 79 n/a 897 1913 1932 524986 TCCAAATTCTTTATAAGGGT 80 n/a 898 1916 1935 524987 AGCTCCAAATTCTTTATAAG 80 n/a 899 1919 1938 524988 AGTAGCTCCAAATTCTTTAT 76 n/a 900 1922 1941 524989 CACAGTAGCTCCAAATTCTT 59 n/a 901 1925 1944 524990 CTCCACAGTAGCTCCAAATT 46 n/a 902 1928 1947 524991 TAACTCCACAGTAGCTCCAA 63 n/a 903 1931 1950 524992 GAGTAACTCCACAGTAGCTC 65 n/a 904 1934 1953 524993 CGAGAGTAACTCCACAGTAG 69 n/a 905 1937 1956 524994 AAACGAGAGTAACTCCACAG 61 n/a 906 1940 1959 524995 CAAAAACGAGAGTAACTCCA 46 n/a 907 1943 1962 524996 AGGCAAAAACGAGAGTAACT 39 n/a 908 1946 1965 524997 AGAAGGCAAAAACGAGAGTA 53 n/a 909 1949 1968 524998 GTCAGAAGGCAAAAACGAGA 56 n/a 910 1952 1971 524999 GAAGTCAGAAGGCAAAAACG 49 n/a 911 1955 1974 525000 AAAGAAGTCAGAAGGCAAAA 29 n/a 912 1958 1977 525001 AGGAAAGAAGTCAGAAGGCA 41 n/a 913 1961 1980 525002 TGAAGGAAAGAAGTCAGAAG 34 n/a 914 1964 1983 525003 TACTGAAGGAAAGAAGTCAG 26 n/a 915 1984 2003 525004 GCGGTATCTAGAAGATCTCG 24 n/a 916 1987 2006 525005 GAGGCGGTATCTAGAAGATC 29 n/a 917 1990 2009 525006 GCTGAGGCGGTATCTAGAAG 29 n/a 918 1993 2012 525007 AGAGCTGAGGCGGTATCTAG 13 n/a 919 1996 2015 525008 TACAGAGCTGAGGCGGTATC 6 n/a 920 1999 2018 525009 CGATACAGAGCTGAGGCGGT 3 n/a 921 2002 2021 525010 TCCCGATACAGAGCTGAGGC 27 n/a 922 2005 2024 525011 GCTTCCCGATACAGAGCTGA 43 n/a 923 2008 2027 525012 AAGGCTTCCCGATACAGAGC 33 n/a 924 2011 2030 525013 TCTAAGGCTTCCCGATACAG 34 n/a 925 2014 2033 525014 GACTCTAAGGCTTCCCGATA 38 n/a 926 2017 2036 525015 GGAGACTCTAAGGCTTCCCG 16 n/a 927 2020 2039 525016 TCAGGAGACTCTAAGGCTTC 16 n/a 928 2023 2042 525017 TGCTCAGGAGACTCTAAGGC 14 n/a 929 2026 2045 525018 CAATGCTCAGGAGACTCTAA 34 n/a 930 2029 2048 525019 GAACAATGCTCAGGAGACTC 32 n/a 931 2032 2051 525020 GGTGAACAATGCTCAGGAGA 9 n/a 932 2035 2054 525021 TGAGGTGAACAATGCTCAGG 50 n/a 933 2038 2057 525022 TGGTGAGGTGAACAATGCTC 54 n/a 934 2041 2060 525023 GTATGGTGAGGTGAACAATG 47 n/a 935 2044 2063 525024 GCAGTATGGTGAGGTGAACA 40 n/a 936 2047 2066 525025 AGTGCAGTATGGTGAGGTGA 35 n/a 937 2050 2069 525026 CTGAGTGCAGTATGGTGAGG 43 n/a 938 2053 2072 525027 TGCCTGAGTGCAGTATGGTG 45 n/a 939 2056 2075 525028 GCTTGCCTGAGTGCAGTATG 42 n/a 940 2059 2078 525029 ATTGCTTGCCTGAGTGCAGT 39 n/a 941 2062 2081 525030 AGAATTGCTTGCCTGAGTGC 27 n/a 942 2065 2084 525031 CAAAGAATTGCTTGCCTGAG 42 n/a 943 2068 2087 525032 CAGCAAAGAATTGCTTGCCT 49 n/a 944 2071 2090 525033 CCCCAGCAAAGAATTGCTTG 41 n/a 945 2074 2093 525034 TCCCCCCAGCAAAGAATTGC 39 n/a 946 2077 2096 525035 AGTTCCCCCCAGCAAAGAAT 39 n/a 947 2080 2099 525036 ATTAGTTCCCCCCAGCAAAG 43 n/a 948 2083 2102 525037 GTCATTAGTTCCCCCCAGCA 64 n/a 949 2086 2105 525038 AGAGTCATTAGTTCCCCCCA 45 n/a 950 2089 2108 525039 GCTAGAGTCATTAGTTCCCC 58 n/a 951 2092 2111 525040 GTAGCTAGAGTCATTAGTTC 45 n/a 952 2095 2114 525041 CAGGTAGCTAGAGTCATTAG 44 n/a 953 2098 2117 525042 ACCCAGGTAGCTAGAGTCAT 39 n/a 954 2101 2120 525043 CCCACCCAGGTAGCTAGAGT 51 n/a 955 2104 2123 525044 ACACCCACCCAGGTAGCTAG 27 n/a 956 2107 2126 525045 TTAACACCCACCCAGGTAGC 41 n/a 957 2110 2129 525046 AAATTAACACCCACCCAGGT 44 n/a 958 2113 2132 525047 TCCAAATTAACACCCACCCA 29 n/a 959 2116 2135 525048 TCTTCCAAATTAACACCCAC 31 n/a 960 2119 2138 525049 GGATCTTCCAAATTAACACC 42 n/a 961 2122 2141 525050 GCTGGATCTTCCAAATTAAC 53 n/a 962 2125 2144 525051 GATGCTGGATCTTCCAAATT 41 n/a 963 2128 2147 525052 CTAGATGCTGGATCTTCCAA 62 n/a 964 2131 2150 525053 TCTCTAGATGCTGGATCTTC 41 83 965 2134 2153 525054 AGGTCTCTAGATGCTGGATC 26 73 966 2137 2156 525055 ACTAGGTCTCTAGATGCTGG 36 74 967 2140 2159 525056 ACTACTAGGTCTCTAGATGC 22 63 968 2143 2162 525057 CTGACTACTAGGTCTCTAGA 28 80 969 2146 2165 525058 TAACTGACTACTAGGTCTCT 47 83 970 2149 2168 525059 ACATAACTGACTACTAGGTC 31 77 971 2152 2171 525060 TTGACATAACTGACTACTAG 34 75 972 2155 2174 525061 GTGTTGACATAACTGACTAC 42 75 973 2158 2177 525062 TTAGTGTTGACATAACTGAC 48 81 974 2161 2180 525063 ATATTAGTGTTGACATAACT 33 73 975 2164 2183 525064 CCCATATTAGTGTTGACATA 41 82 976 2167 2186 525065 AGGCCCATATTAGTGTTGAC 39 77 977 2170 2189 525066 TTTAGGCCCATATTAGTGTT 46 83 978 2173 2192 525067 AACTTTAGGCCCATATTAGT 38 69 979 2176 2195 525068 CTGAACTTTAGGCCCATATT 41 85 980 2179 2198 525069 TGCCTGAACTTTAGGCCCAT 38 81 981 2182 2201 525070 AGTTGCCTGAACTTTAGGCC 17 67 982 2185 2204 525071 AAGAGTTGCCTGAACTTTAG 27 62 983 2188 2207 525072 CACAAGAGTTGCCTGAACTT 27 64 984 2191 2210 525073 AACCACAAGAGTTGCCTGAA 41 80 985 2194 2213 525074 TGAAACCACAAGAGTTGCCT 32 75 986 2197 2216 525075 ATGTGAAACCACAAGAGTTG 43 67 987 2200 2219 525076 GAAATGTGAAACCACAAGAG 34 74 988 2203 2222 525077 CAAGAAATGTGAAACCACAA 22 65 989 2206 2225 525078 AGACAAGAAATGTGAAACCA 39 70 990 2209 2228 525079 GTGAGACAAGAAATGTGAAA 32 74 991 2212 2231 525080 AAAGTGAGACAAGAAATGTG 30 63 992 2215 2234 525081 CCAAAAGTGAGACAAGAAAT 25 58 993 2218 2237 525082 CTTCCAAAAGTGAGACAAGA 36 74 994 2221 2240 525083 TCTCTTCCAAAAGTGAGACA 42 84 995 2224 2243 525084 GTTTCTCTTCCAAAAGTGAG 33 75 996 2227 2246 525085 ACGGTTTCTCTTCCAAAAGT 32 68 997 2230 2249 525086 ATAACGGTTTCTCTTCCAAA 51 80 998 2233 2252 525087 TCTATAACGGTTTCTCTTCC 36 77 999 2236 2255 525088 TACTCTATAACGGTTTCTCT 23 69 1000 2239 2258 525089 AAATACTCTATAACGGTTTC 45 77 1001 2242 2261 525090 ACCAAATACTCTATAACGGT 57 82 1002 2245 2264 525091 GACACCAAATACTCTATAAC 36 77 1003 2248 2267 525092 AAAGACACCAAATACTCTAT 42 80 1004 2251 2270 525093 CCGAAAGACACCAAATACTC 41 89 1005 2254 2273 525094 ACTCCGAAAGACACCAAATA 29 73 1006 2257 2276 525095 CACACTCCGAAAGACACCAA 33 92 1007 2260 2279 525096 ATCCACACTCCGAAAGACAC 18 74 1008 2263 2282 525097 CGAATCCACACTCCGAAAGA 30 57 1009 2266 2285 525098 GTGCGAATCCACACTCCGAA 28 67 1010 2269 2288 146789 GGAGTGCGAATCCACACTCC 37 72 1011 2272 2291 525099 GGAGGAGTGCGAATCCACAC 36 64 1012 2275 2294 525100 GCTGGAGGAGTGCGAATCCA 52 90 1013 2278 2297 525101 TAAGCTGGAGGAGTGCGAAT 49 96 1014 2281 2300 525102 CTATAAGCTGGAGGAGTGCG 37 96 1015 2284 2303 525103 GGTCTATAAGCTGGAGGAGT 30 97 1016 2287 2306 525104 GGTGGTCTATAAGCTGGAGG 22 77 1017 2290 2309 525105 TTTGGTGGTCTATAAGCTGG 41 76 1018 2293 2312 525106 GCATTTGGTGGTCTATAAGC 39 76 1019 2313 2332 525107 GAAGTGTTGATAGGATAGGG 27 97 1020 2316 2335 525108 CCGGAAGTGTTGATAGGATA 42 97 1021 2319 2338 525109 TTTCCGGAAGTGTTGATAGG 48 99 1022 2322 2341 525110 TAGTTTCCGGAAGTGTTGAT 18 98 1023 2325 2344 525111 CAGTAGTTTCCGGAAGTGTT 19 98 1024 2328 2347 525112 CAACAGTAGTTTCCGGAAGT 29 96 1025 2331 2350 525113 TAACAACAGTAGTTTCCGGA 39 95 1026 2334 2353 525114 GTCTAACAACAGTAGTTTCC 40 99 1027 2369 2388 525115 CGAGGGAGTTCTTCTTCTAG 42 98 1028 2372 2391 525116 AGGCGAGGGAGTTCTTCTTC 31 97 1029 2375 2394 525117 GCGAGGCGAGGGAGTTCTTC 22 98 1030 2379 2398 525118 GTCTGCGAGGCGAGGGAGTT 20 99 1031 2398 2417 525119 CGCGGCGATTGAGACCTTCG 26 97 1032 2401 2420 525120 CGACGCGGCGATTGAGACCT 23 97 1033 2404 2423 525121 CTGCGACGCGGCGATTGAGA 47 92 1034 2407 2426 525122 CTTCTGCGACGCGGCGATTG 27 74 1035 2410 2429 525123 GATCTTCTGCGACGCGGCGA 36 87 1036 2413 2432 146790 TGAGATCTTCTGCGACGCGG 25 85 1037 2416 2435 525124 GATTGAGATCTTCTGCGACG 17 84 1038 2419 2438 525125 CGAGATTGAGATCTTCTGCG 24 82 1039 2422 2441 525126 TCCCGAGATTGAGATCTTCT 29 74 1040 2425 2444 525127 GGTTCCCGAGATTGAGATCT 14 79 1041 2428 2447 525128 TGAGGTTCCCGAGATTGAGA 41 76 1042 2431 2450 525129 CATTGAGGTTCCCGAGATTG 39 72 1043 2434 2453 525130 TAACATTGAGGTTCCCGAGA 37 71 1044 2437 2456 525131 TACTAACATTGAGGTTCCCG 42 76 1045 2440 2459 525132 GAATACTAACATTGAGGTTC 21 75 1046 2443 2462 525133 AAGGAATACTAACATTGAGG 36 75 1047 2446 2465 525134 TCCAAGGAATACTAACATTG 29 77 1048 2449 2468 525135 GAGTCCAAGGAATACTAACA 32 76 1049 2452 2471 525136 TATGAGTCCAAGGAATACTA 23 62 1050 2455 2474 525137 CCTTATGAGTCCAAGGAATA 27 57 1051 2458 2477 525138 CCACCTTATGAGTCCAAGGA 52 82 1052 2461 2480 525139 TCCCCACCTTATGAGTCCAA 46 80 1053 2464 2483 525140 AGTTCCCCACCTTATGAGTC 14 59 1054 2467 2486 525141 TAAAGTTCCCCACCTTATGA 20 45 1055 2470 2489 525142 CAGTAAAGTTCCCCACCTTA 14 72 1056 2473 2492 525143 GACCAGTAAAGTTCCCCACC 30 77 1057 2476 2495 525144 AAAGACCAGTAAAGTTCCCC 19 72 1058 2479 2498 525145 AATAAAGACCAGTAAAGTTC 18 55 1059 2482 2501 525146 AAGAATAAAGACCAGTAAAG 16 51 1060 2485 2504 525147 TAGAAGAATAAAGACCAGTA 22 68 1061 2488 2507 525148 CAGTAGAAGAATAAAGACCA 13 59 1062 2491 2510 525149 GTACAGTAGAAGAATAAAGA 0 45 1063 2494 2513 525150 CAGGTACAGTAGAAGAATAA 31 62 1064 2497 2516 525151 AGACAGGTACAGTAGAAGAA 8 62 1065 2500 2519 525152 TAAAGACAGGTACAGTAGAA 29 61 1066 2503 2522 525153 GATTAAAGACAGGTACAGTA 28 67 1067 2506 2525 525154 GAGGATTAAAGACAGGTACA 38 76 1068 2509 2528 525155 AATGAGGATTAAAGACAGGT 30 72 1069 2512 2531 525156 TCCAATGAGGATTAAAGACA 24 67 1070 2515 2534 525157 TTTTCCAATGAGGATTAAAG 0 44 1071 2518 2537 525158 GTGTTTTCCAATGAGGATTA 20 74 1072 2521 2540 525159 ATGGTGTTTTCCAATGAGGA 30 71 1073 2524 2543 525160 AAGATGGTGTTTTCCAATGA 22 68 1074 2527 2546 525161 GAAAAGATGGTGTTTTCCAA 19 61 1075 2530 2549 525162 TAGGAAAAGATGGTGTTTTC 14 52 1076 2533 2552 525163 TATTAGGAAAAGATGGTGTT 1 47 1077 2536 2555 525164 GTATATTAGGAAAAGATGGT 0 60 1078 2539 2558 525165 AATGTATATTAGGAAAAGAT 0 30 1079 2542 2561 525166 GTAAATGTATATTAGGAAAA 1 18 1080 2545 2564 525167 GGTGTAAATGTATATTAGGA 23 72 1081 2548 2567 525168 CTTGGTGTAAATGTATATTA 32 75 1082 2551 2570 525169 TGTCTTGGTGTAAATGTATA 12 65 1083 2554 2573 525170 TAATGTCTTGGTGTAAATGT 3 51 1084 2557 2576 525171 TGATAATGTCTTGGTGTAAA 24 62 1085 2560 2579 525172 TTTTGATAATGTCTTGGTGT 18 66 1086 2563 2582 525173 ATTTTTTGATAATGTCTTGG 11 63 1087 2566 2585 525174 CACATTTTTTGATAATGTCT 20 68 1088 2569 2588 525175 GTTCACATTTTTTGATAATG 38 68 1089 2572 2591 525176 ACTGTTCACATTTTTTGATA 12 61 1090 2575 2594 525177 CAAACTGTTCACATTTTTTG 25 56 1091 2578 2597 525178 CTACAAACTGTTCACATTTT 21 47 1092 2581 2600 525179 GGCCTACAAACTGTTCACAT 28 83 1093 2584 2603 525180 GTGGGCCTACAAACTGTTCA 7 72 1094 2587 2606 525181 TAAGTGGGCCTACAAACTGT 26 75 1095 2590 2609 525182 CTGTAAGTGGGCCTACAAAC 35 78 1096 2593 2612 525183 TAACTGTAAGTGGGCCTACA 29 69 1097 2596 2615 525184 CATTAACTGTAAGTGGGCCT 22 73 1098 2599 2618 525185 TCTCATTAACTGTAAGTGGG 31 81 1099 2602 2621 525186 TTTTCTCATTAACTGTAAGT 15 58 1100 2605 2624 525187 TTCTTTTCTCATTAACTGTA 14 71 1101 2608 2627 525188 ATCTTCTTTTCTCATTAACT 19 71 1102 2611 2630 525189 GCAATCTTCTTTTCTCATTA 36 79 1103 2614 2633 525190 ATTGCAATCTTCTTTTCTCA 38 82 1104 2617 2636 525191 TCAATTGCAATCTTCTTTTC 23 61 1105 2620 2639 525192 TAATCAATTGCAATCTTCTT 10 67 1106 2623 2642 525193 GCATAATCAATTGCAATCTT 27 71 1107 2626 2645 525194 CAGGCATAATCAATTGCAAT 23 71 1108 2629 2648 525195 TAGCAGGCATAATCAATTGC 30 77 1109 2632 2651 525196 ACCTAGCAGGCATAATCAAT 7 70 1110 2635 2654 525197 AAAACCTAGCAGGCATAATC 47 70 1111 2638 2657 525198 GATAAAACCTAGCAGGCATA 41 81 1112 2641 2660 525199 TTGGATAAAACCTAGCAGGC 30 78 1113 2644 2663 525200 CCTTTGGATAAAACCTAGCA 31 76 1114 2647 2666 525201 TAACCTTTGGATAAAACCTA 25 63 1115 2650 2669 525202 TGGTAACCTTTGGATAAAAC 24 76 1116 2653 2672 525203 ATTTGGTAACCTTTGGATAA 20 64 1117 2656 2675 525204 AATATTTGGTAACCTTTGGA 16 77 1118 2659 2678 525205 GTAAATATTTGGTAACCTTT 39 80 1119 2662 2681 525206 ATGGTAAATATTTGGTAACC 40 75 1120 2665 2684 525207 CCAATGGTAAATATTTGGTA 38 75 1121 2668 2687 525208 TATCCAATGGTAAATATTTG 0 0 1122 2671 2690 525209 CCTTATCCAATGGTAAATAT 28 57 1123 2674 2693 525210 TACCCTTATCCAATGGTAAA 18 71 1124 2677 2696 525211 TAATACCCTTATCCAATGGT 35 76 1125 2680 2699 525212 GTTTAATACCCTTATCCAAT 41 77 1126 2683 2702 525213 AAGGTTTAATACCCTTATCC 11 79 1127 2686 2705 525214 AATAAGGTTTAATACCCTTA 35 75 1128 2689 2708 525215 GATAATAAGGTTTAATACCC 22 54 1129 2692 2711 525216 CTGGATAATAAGGTTTAATA 19 35 1130 2695 2714 525217 GTTCTGGATAATAAGGTTTA 24 58 1131 2698 2717 525218 GATGTTCTGGATAATAAGGT 20 73 1132 2701 2720 525219 CTAGATGTTCTGGATAATAA 26 66 1133 2704 2723 525220 TAACTAGATGTTCTGGATAA 21 66 1134 2707 2726 525221 GATTAACTAGATGTTCTGGA 30 78 1135 2710 2729 525222 AATGATTAACTAGATGTTCT 30 61 1136 2713 2732 525223 AGTAATGATTAACTAGATGT 9 57 1137 2716 2735 525224 GGAAGTAATGATTAACTAGA 18 72 1138 2719 2738 525225 TTTGGAAGTAATGATTAACT 7 67 1139 2722 2741 525226 TAGTTTGGAAGTAATGATTA 2 30 1140 2725 2744 525227 GTCTAGTTTGGAAGTAATGA 27 78 1141 2728 2747 525228 AGTGTCTAGTTTGGAAGTAA 27 75 1142 2731 2750 525229 AATAGTGTCTAGTTTGGAAG 34 73 1143 2734 2753 525230 GTAAATAGTGTCTAGTTTGG 28 68 1144 2737 2756 525231 TGTGTAAATAGTGTCTAGTT 27 79 1145 2740 2759 525232 GAGTGTGTAAATAGTGTCTA 27 71 1146 2743 2762 525233 ATAGAGTGTGTAAATAGTGT 17 75 1147 2746 2765 525234 TCCATAGAGTGTGTAAATAG 18 75 1148 2749 2768 525235 CCTTCCATAGAGTGTGTAAA 23 80 1149 2752 2771 525236 CCGCCTTCCATAGAGTGTGT 26 82 1150 2755 2774 525237 TACCCGCCTTCCATAGAGTG 19 80 1151 2758 2777 525238 ATATACCCGCCTTCCATAGA 0 67 1152 2761 2780 525239 ATAATATACCCGCCTTCCAT 19 70 1153 2764 2783 525240 TATATAATATACCCGCCTTC 9 73 1154 2767 2786 525241 TCTTATATAATATACCCGCC 20 80 1155 2770 2789 525242 CTCTCTTATATAATATACCC 29 76 1156 2773 2792 525243 TTTCTCTCTTATATAATATA 16 58 1157 2776 2795 525244 TTGTTTCTCTCTTATATAAT 26 57 1158 2779 2798 525245 GTGTTGTTTCTCTCTTATAT 35 85 1159 2782 2801 525246 TATGTGTTGTTTCTCTCTTA 34 82 1160 2785 2804 525247 CGCTATGTGTTGTTTCTCTC 34 86 1161 2802 2821 525248 TGACCCACAAAATGAGGCGC 17 71 1162 2805 2824 525249 TGGTGACCCACAAAATGAGG 31 67 1163 2808 2827 525250 ATATGGTGACCCACAAAATG 38 69 1164 2811 2830 525251 AGAATATGGTGACCCACAAA 37 77 1165 2814 2833 525252 CCAAGAATATGGTGACCCAC 35 79 1166 2817 2836 146831 TTCCCAAGAATATGGTGACC 27 75 1167 2820 2839 525253 TTGTTCCCAAGAATATGGTG 33 69 1168 2823 2842 525254 ATCTTGTTCCCAAGAATATG 27 65 1169 2826 2845 525255 TAGATCTTGTTCCCAAGAAT 31 70 1170 2829 2848 525256 CTGTAGATCTTGTTCCCAAG 42 81 1171 2832 2851 525257 ATGCTGTAGATCTTGTTCCC 34 80 1172 2835 2854 525258 CCCATGCTGTAGATCTTGTT 38 80 1173 2838 2857 525259 TGCCCCATGCTGTAGATCTT 36 80 1174 2841 2860 525260 TTCTGCCCCATGCTGTAGAT 32 74 1175 2844 2863 525261 AGATTCTGCCCCATGCTGTA 27 75 1176 2847 2866 525262 GAAAGATTCTGCCCCATGCT 34 70 1177 2850 2869 525263 GTGGAAAGATTCTGCCCCAT 22 76 1178 2853 2872 525264 CTGGTGGAAAGATTCTGCCC 36 72 1179 2856 2875 525265 TTGCTGGTGGAAAGATTCTG 32 71 1180 2859 2878 525266 GGATTGCTGGTGGAAAGATT 20 74 1181 2862 2881 525267 AGAGGATTGCTGGTGGAAAG 25 73 1182 2865 2884 525268 CCCAGAGGATTGCTGGTGGA 40 82 1183 2868 2887 525269 AATCCCAGAGGATTGCTGGT 32 79 1184 2871 2890 525270 AAGAATCCCAGAGGATTGCT 23 69 1185 2874 2893 525271 GGAAAGAATCCCAGAGGATT 10 66 1186 2877 2896 525272 TCGGGAAAGAATCCCAGAGG 29 73 1187 2880 2899 525273 TGGTCGGGAAAGAATCCCAG 31 77 1188 2883 2902 525274 TGGTGGTCGGGAAAGAATCC 38 71 1189 2886 2905 525275 AACTGGTGGTCGGGAAAGAA 33 78 1190 2889 2908 525276 TCCAACTGGTGGTCGGGAAA 29 76 1191 2892 2911 525277 GGATCCAACTGGTGGTCGGG 19 81 1192 2895 2914 525278 GCTGGATCCAACTGGTGGTC 24 74 1193 2898 2917 525279 AAGGCTGGATCCAACTGGTG 33 83 1194 2901 2920 525280 CTGAAGGCTGGATCCAACTG 18 81 1195 2904 2923 525286 GCTCTGAAGGCTGGATCCAA 40 79 1196 2907 2926 525287 TTTGCTCTGAAGGCTGGATC 34 69 1197 2910 2929 525288 GTGTTTGCTCTGAAGGCTGG 38 72 1198 2913 2932 525289 GCTGTGTTTGCTCTGAAGGC 40 82 1199 2916 2935 525290 TTTGCTGTGTTTGCTCTGAA 44 78 1200 2919 2938 525291 GGATTTGCTGTGTTTGCTCT 38 76 1201 2922 2941 525292 TCTGGATTTGCTGTGTTTGC 28 79 1202 2925 2944 525293 CAATCTGGATTTGCTGTGTT 26 61 1203 2928 2947 525294 TCCCAATCTGGATTTGCTGT 32 68 1204 2931 2950 525295 AAGTCCCAATCTGGATTTGC 33 59 1205 2934 2953 146832 TTGAAGTCCCAATCTGGATT 17 35 1206 2937 2956 525296 GGATTGAAGTCCCAATCTGG 35 62 1207 2940 2959 525297 TTGGGATTGAAGTCCCAATC 10 36 1208 2943 2962 525298 TTGTTGGGATTGAAGTCCCA 24 49 1209 2946 2965 525299 TCCTTGTTGGGATTGAAGTC 16 52 1210 2949 2968 525300 GTGTCCTTGTTGGGATTGAA 18 71 1211 2952 2971 525301 CAGGTGTCCTTGTTGGGATT 25 73 1212 2955 2974 525302 GGCCAGGTGTCCTTGTTGGG 31 70 1213 2958 2977 525303 TCTGGCCAGGTGTCCTTGTT 29 75 1214 2978 2997 525304 CAGCTCCTACCTTGTTGGCG 29 71 1215 2981 3000 525305 CTCCAGCTCCTACCTTGTTG 19 63 1216 2984 3003 525306 ATGCTCCAGCTCCTACCTTG 35 75 1217 2987 3006 525307 CGAATGCTCCAGCTCCTACC 13 77 1218 2990 3009 525308 GCCCGAATGCTCCAGCTCCT 28 72 1219 2993 3012 525309 CCAGCCCGAATGCTCCAGCT 32 77 1220 2996 3015 525310 AACCCAGCCCGAATGCTCCA 34 72 1221 2999 3018 525311 TGAAACCCAGCCCGAATGCT 28 69 1222 3002 3021 525312 GGGTGAAACCCAGCCCGAAT 18 68 1223 3020 3039 525313 AAAGGCCTCCGTGCGGTGGG 36 77 1224 3023 3042 525314 CCAAAAGGCCTCCGTGCGGT 34 83 1225 3026 3045 525315 ACCCCAAAAGGCCTCCGTGC 28 70 1226 3029 3048 525316 TCCACCCCAAAAGGCCTCCG 26 65 1227 3032 3051 525317 GGCTCCACCCCAAAAGGCCT 19 36 1228 3035 3054 525318 GAGGGCTCCACCCCAAAAGG 14 36 1229 3038 3057 525319 CCTGAGGGCTCCACCCCAAA 32 71 1230 3041 3060 525320 GAGCCTGAGGGCTCCACCCC 37 61 1231 3044 3063 525321 CCTGAGCCTGAGGGCTCCAC 42 70 1232 3047 3066 525322 TGCCCTGAGCCTGAGGGCTC 24 56 1233 3050 3069 525323 GTATGCCCTGAGCCTGAGGG 14 75 1234 3053 3072 525324 GTAGTATGCCCTGAGCCTGA 29 83 1235 3056 3075 525325 TTTGTAGTATGCCCTGAGCC 32 61 1236 3059 3078 525326 AAGTTTGTAGTATGCCCTGA 35 70 1237 3062 3081 525327 GCAAAGTTTGTAGTATGCCC 37 61 1238 3065 3084 525328 CTGGCAAAGTTTGTAGTATG 26 63 1239 3068 3087 525329 TTGCTGGCAAAGTTTGTAGT 37 74 1240 3071 3090 525330 GATTTGCTGGCAAAGTTTGT 20 56 1241 3074 3093 525331 GCGGATTTGCTGGCAAAGTT 28 80 1242 3077 3096 525332 GAGGCGGATTTGCTGGCAAA 38 74 1243 3080 3099 525333 CAGGAGGCGGATTTGCTGGC 41 66 1244 3083 3102 525334 AGGCAGGAGGCGGATTTGCT 27 55 1245 3086 3105 525335 TGGAGGCAGGAGGCGGATTT 13 17 1246 3089 3108 525336 TGGTGGAGGCAGGAGGCGGA 7 21 1247 3092 3111 525337 GATTGGTGGAGGCAGGAGGC 21 44 1248 3095 3114 525338 GGCGATTGGTGGAGGCAGGA 31 65 1249 3098 3117 525339 TCTGGCGATTGGTGGAGGCA 15 76 1250 3101 3120 525340 CTGTCTGGCGATTGGTGGAG 35 73 1251 3104 3123 525341 TTCCTGTCTGGCGATTGGTG 32 72 1252 3107 3126 525342 GCCTTCCTGTCTGGCGATTG 28 64 1253 3110 3129 525343 GCTGCCTTCCTGTCTGGCGA 25 69 1254 3113 3132 525344 TAGGCTGCCTTCCTGTCTGG 32 79 1255 3116 3135 525345 GGGTAGGCTGCCTTCCTGTC 35 80 1256 3134 3153 525346 TCAAAGGTGGAGACAGCGGG 4 57 1257 3137 3156 525347 TTCTCAAAGGTGGAGACAGC 32 72 1258 3140 3159 525348 TGTTTCTCAAAGGTGGAGAC 32 66 1259 3143 3162 525349 GAGTGTTTCTCAAAGGTGGA 34 63 1260 3146 3165 525350 GATGAGTGTTTCTCAAAGGT 35 68 1261 3149 3168 525351 GAGGATGAGTGTTTCTCAAA 36 84 1262 3152 3171 525352 CCTGAGGATGAGTGTTTCTC 44 77 1263 3155 3174 525353 TGGCCTGAGGATGAGTGTTT 32 72 1264 3158 3177 525354 GCATGGCCTGAGGATGAGTG 27 73 1265 3162 3181 525355 CACTGCATGGCCTGAGGATG 40 69 1266

TABLE 25 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides targeted to SEQ ID NO: 1286 (RTS3370 and RTS3372) Viral Viral Start Stop RTS3370 % RTS3372 % SEQ ID Site Site ISIS No Sequence inhibition inhibition NO 85 104 525356 TTCCACTGCATGGCCTGAGG 53 78 1267 88 107 525357 GAATTCCACTGCATGGCCTG 44 68 1268 91 110 525358 GTGGAATTCCACTGCATGGC 42 80 1269 94 113 525359 GTTGTGGAATTCCACTGCAT 45 77 1270 97 116 525360 AAGGTTGTGGAATTCCACTG 65 67 1271 100 119 525361 TGAAAGGTTGTGGAATTCCA 56 61 1272

Example 12 Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers

Certain gapmers from the study described in Examples 11 and 12 were tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE 2000® reagent with 5.56 nM, 16.67 nM, 50.0 nM, and 150.0 nM concentrations of antisense oligonucleotide, as specified in Table 26. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 26. As illustrated in Table 26, HBV mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 26 Dose-dependent antisense inhibition of HBV RNA in HepG2.2.15 cells using RTS3370 IC₅₀ ISIS No 5.5556 nM 16.6667 nM 50.0 nM 150.0 nM (nM) 146785 0 0 14 66 120.8 146786 40 64 78 88 8.5 505314 23 35 58 84 28.8 505339 28 42 62 84 23.2 505347 9 21 45 75 53.5 514469 11 22 69 79 35.4 524493 13 39 56 81 32.8 524540 15 38 54 80 34.0 524617 14 32 78 83 27.1 524619 33 42 60 84 21.3 524634 20 45 63 80 26.3 524641 39 49 62 86 14.9 524698 34 34 49 64 47.4 524699 25 31 44 63 66.1 524706 29 20 36 58 128.8 524709 32 26 48 56 89.1 524718 46 41 61 79 15.8 524731 49 53 68 83 8.1 524734 42 31 35 64 87.2 524767 19 38 62 84 27.8 524768 35 38 62 75 23.5 524769 16 26 61 75 38.1 524806 0 0 0 35 >150.0 524807 3 22 39 74 60.2 524907 22 35 63 80 29.1 524908 25 45 67 78 22.9 524976 7 3 0 16 >150.0 524978 6 0 0 27 >150.0 524979 3 0 11 34 >150.0 524980 18 51 48 59 51.5 524981 16 27 49 61 65.8 524982 21 19 29 54 >150.0 524983 23 40 50 60 53.2 524984 19 25 45 74 50.0 524985 13 19 40 56 107.2 524986 29 48 46 64 39.3 524987 17 0 43 61 102.8 524988 22 39 52 63 47.6 524991 0 7 19 20 >150.0 524997 17 0 1 9 >150.0 524998 1 5 8 34 >150.0 525095 5 0 0 18 >150.0 525100 14 5 14 26 >150.0 525101 0 0 15 19 >150.0 525102 0 0 18 23 >150.0 525103 0 0 3 15 >150.0 525179 18 7 9 18 >150.0 525245 0 0 8 8 >150.0 525247 12 15 16 23 >150.0 525289 1 1 15 30 >150.0 525314 17 0 18 25 >150.0 525324 0 6 13 16 >150.0 525351 28 13 22 30 >150.0

Some of the ISIS-oligonucleotides were also tested using primer probe set RTS3372. The results are presented in Table 27.

TABLE 27 Dose-dependent antisense inhibition of HBV RNA in HepG2.2.15 cells using RTS3372 IC₅₀ ISIS No 5.5556 nM 16.6667 nM 50.0 nM 150.0 nM (nM) 146785 0 0 0 51 >150.0 146786 41 68 81 91 7.9 505347 0 13 44 75 59.7 524103 0 0 1 9 >150.0 524245 0 0 6 10 >150.0 524767 18 46 60 85 25.8 524768 34 41 66 79 20.5 524769 12 38 60 77 34.5 524806 0 0 0 0 >150.0 524807 0 9 34 70 78.6 524907 20 41 62 84 26.4 524908 27 45 66 82 21.3 524976 0 0 0 16 >150.0 524978 3 0 0 22 >150.0 524979 0 0 0 33 >150.0 524980 28 51 52 67 30.1 524981 7 29 51 66 55.8 524982 22 29 37 63 83.5 524983 20 51 43 62 50.9 524984 20 30 38 75 51.7 524985 30 33 40 60 83.6 524986 25 51 51 66 33.8 524987 19 0 24 65 157.6 524988 12 41 45 62 59.2 524991 0 0 4 8 >150.0 524997 19 0 0 15 >150.0 524998 0 0 1 42 >150.0 525095 0 0 0 17 >150.0 525100 10 0 4 19 >150.0 525101 10 0 21 25 >150.0 525102 0 0 10 15 >150.0 525247 11 12 15 28 >150.0 525289 0 9 11 33 >150.0 525314 1 0 18 24 >150.0 525324 9 8 15 10 >150.0

Example 13 Dose-Dependent Inhibition of Viral HBV RNA in HepG2.2.15 Cells by MOE Gapmers

Certain gapmers from the studies described above were tested at various doses in human HepG2.2.15 cells. Cells were plated at a density of 30,000 cells per well and transfected using LipofectAMINE 2000® reagent with 7.8125 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125.0 nM and 250.0 nM concentrations of antisense oligonucleotide, as specified in Table 28. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 28. As illustrated in Table 28, HBV mRNA levels were reduced in a dose-dependent manner in several antisense oligonucleotide treated cells.

TABLE 28 Dose-dependent antisense inhibition of HBV RNA in HepG2.2.15 cells using RTS3370 ISIS 7.8125 15.625 31.25 62.5 125.0 250.0 IC₅₀ No nM nM nM nM nM nM (nM) 146786 0 0 14 49 33 50 161.2 510100 0 17 30 28 44 53 177.8 510106 0 4 0 0 29 0 >250.0 509934 0 0 0 7 16 0 >250.0 510116 0 0 8 21 27 25 >250.0 505347 31 3 30 63 80 81 48.7

Example 14 Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables. ISIS 146786, 509934, ISIS 509959, and ISIS 510100, from the studies described above, were also included. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 2; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 3; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 4) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 311; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 312; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 313). HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells. In some of the assays shown in Tables 32, 35, 42, 45, and 46, the potency of ISIS 146786 was measured in two wells in a single plate. In those cases, the values of inhibition levels in both wells have been presented.

The newly designed chimeric antisense oligonucleotides in Tables below were designed as either 2-9-5 MOE gapmers, 2-9-6 MOE gapmers, 2-10-8 MOE gapmers, 3-9-4 MOE gapmers, 3-9-5 MOE gapmers, 3-10-3 MOE gapmers, 3-10-4 MOE gapmers, 3-10-7 MOE gapmers, 4-9-3 MOE gapmers, 4-9-4 MOE gapmers, 4-10-6 MOE gapmers, 5-9-2 MOE gapmers, 5-9-3 MOE gapmers, 5-10-5 MOE gapmers, 6-9-2 MOE gapmers, 6-10-4 MOE gapmers, 7-10-3 MOE gapmers, or 8-10-2 MOE gapmers. The 2-9-5 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two and five nucleosides respectively. The 2-9-6 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two and six nucleosides respectively. The 2-10-8 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two and eight nucleosides respectively. The 3-9-4 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and four nucleosides respectively. The 3-9-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and five nucleosides respectively. The 3-10-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and four nucleosides respectively. The 3-10-7 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three and seven nucleosides respectively. The 4-9-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four and three nucleosides respectively. The 4-9-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four nucleosides each. The 4-10-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four and six nucleosides respectively. The 5-9-2 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five and two nucleosides respectively. The 5-9-3 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five and three nucleosides respectively. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The 6-9-2 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising six and two nucleosides respectively. The 6-10-4 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising six and four nucleosides respectively. The 7-10-3 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising seven and three nucleosides respectively. The 8-10-2 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising eight and two nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has an MOE sugar modification. The ‘Motif’ column indicates the motifs with the number of nucleosides in the wings and the gap segment of each of the oligonucleotides. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. The ‘Motif’ column indicates the gap and wing structure of each gapmer. Each gapmer listed in the Tables is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). The potency of the newly designed oligonucleotides was compared with ISIS 146786, 509934, ISIS 509959, and ISIS 510100, the information of which have been placed at the top of each table.

TABLE 29 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 50 224 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 62 17 58 74 CTGGAGCCACCAGCAGG 552276 5-9-3 42 1288 59 75 ACTGGAGCCACCAGCAG 552277 5-9-3 46 1289 60 76 AACTGGAGCCACCAGCA 552278 5-9-3 31 1290 61 77 GAACTGGAGCCACCAGC 552279 5-9-3 41 1291 253 269 GAAGTCCACCACGAGTC 552280 5-9-3 5 9 254 270 AGAAGTCCACCACGAGT 552281 5-9-3 11 10 255 271 GAGAAGTCCACCACGAG 552282 5-9-3 20 11 256 272 AGAGAAGTCCACCACGA 552283 5-9-3 28 12 411 427 GGCATAGCAGCAGGATG 552230 4-9-4 57 17 411 427 GGCATAGCAGCAGGATG 552284 5-9-3 0 17 412 428 AGGCATAGCAGCAGGAT 552231 4-9-4 29 18 412 428 AGGCATAGCAGCAGGAT 552285 5-9-3 61 18 413 429 GAGGCATAGCAGCAGGA 552232 4-9-4 35 19 413 429 GAGGCATAGCAGCAGGA 552286 5-9-3 47 19 414 430 TGAGGCATAGCAGCAGG 552233 4-9-4 38 21 414 430 TGAGGCATAGCAGCAGG 552287 5-9-3 45 21 415 431 ATGAGGCATAGCAGCAG 552234 4-9-4 0 23 415 431 ATGAGGCATAGCAGCAG 552288 5-9-3 50 23 416 432 GATGAGGCATAGCAGCA 552235 4-9-4 0 25 416 432 GATGAGGCATAGCAGCA 552289 5-9-3 46 25 417 433 AGATGAGGCATAGCAGC 552236 4-9-4 45 27 417 433 AGATGAGGCATAGCAGC 552290 5-9-3 41 27 418 434 AAGATGAGGCATAGCAG 552237 4-9-4 44 29 418 434 AAGATGAGGCATAGCAG 552291 5-9-3 26 29 670 686 ACTAGTAAACTGAGCCA 552239 4-9-4 62 1292 670 686 ACTAGTAAACTGAGCCA 552293 5-9-3 67 1292 671 687 CACTAGTAAACTGAGCC 552240 4-9-4 61 1293 671 687 CACTAGTAAACTGAGCC 552294 5-9-3 71 1293 672 688 GCACTAGTAAACTGAGC 552241 4-9-4 55 1294 672 688 GCACTAGTAAACTGAGC 552295 5-9-3 58 1294 687 703 ACCACTGAACAAATGGC 552242 4-9-4 60 40 687 703 ACCACTGAACAAATGGC 552296 5-9-3 59 40 688 704 AACCACTGAACAAATGG 552243 4-9-4 57 41 688 704 AACCACTGAACAAATGG 552297 5-9-3 55 41 689 705 GAACCACTGAACAAATG 552244 4-9-4 33 42 689 705 GAACCACTGAACAAATG 552298 5-9-3 48 42 690 706 CGAACCACTGAACAAAT 552245 4-9-4 48 43 690 706 CGAACCACTGAACAAAT 552299 5-9-3 34 43 1261 1277 CGCAGTATGGATCGGCA 552246 4-9-4 81 1295 1261 1277 CGCAGTATGGATCGGCA 552300 5-9-3 56 1295 1262 1278 CCGCAGTATGGATCGGC 552247 4-9-4 87 1296 1262 1278 CCGCAGTATGGATCGGC 552301 5-9-3 86 1296 1263 1279 TCCGCAGTATGGATCGG 552248 4-9-4 72 1297 1263 1279 TCCGCAGTATGGATCGG 552302 5-9-3 77 1297 1264 1280 TTCCGCAGTATGGATCG 552249 4-9-4 56 1298 1264 1280 TTCCGCAGTATGGATCG 552303 5-9-3 65 1298 1265 1281 GTTCCGCAGTATGGATC 552250 4-9-4 52 1299 1265 1281 GTTCCGCAGTATGGATC 552304 5-9-3 57 1299 1266 1282 AGTTCCGCAGTATGGAT 552251 4-9-4 43 1300 1266 1282 AGTTCCGCAGTATGGAT 552305 5-9-3 56 1300 1267 1283 GAGTTCCGCAGTATGGA 552252 4-9-4 62 1301 1267 1283 GAGTTCCGCAGTATGGA 552306 5-9-3 75 1301 1268 1284 GGAGTTCCGCAGTATGG 552253 4-9-4 82 1302 1268 1284 GGAGTTCCGCAGTATGG 552307 5-9-3 90 1302 1269 1285 AGGAGTTCCGCAGTATG 552254 4-9-4 74 1303 1577 1593 AAGCGAAGTGCACACGG 552255 4-9-4 78 1304 1578 1594 GAAGCGAAGTGCACACG 552256 4-9-4 65 1305 1579 1595 TGAAGCGAAGTGCACAC 552257 4-9-4 62 1306 1580 1596 GTGAAGCGAAGTGCACA 552258 4-9-4 72 1307 1581 1597 GGTGAAGCGAAGTGCAC 552259 4-9-4 63 1308 1582 1598 AGGTGAAGCGAAGTGCA 552260 4-9-4 58 1309 1583 1599 GAGGTGAAGCGAAGTGC 552261 4-9-4 63 1310 1584 1600 AGAGGTGAAGCGAAGTG 552262 4-9-4 50 1311 1585 1601 CAGAGGTGAAGCGAAGT 552263 4-9-4 60 1312 1586 1602 GCAGAGGTGAAGCGAAG 552264 4-9-4 52 1313 1587 1603 TGCAGAGGTGAAGCGAA 552265 4-9-4 68 1314 1588 1604 GTGCAGAGGTGAAGCGA 552266 4-9-4 62 1315 1589 1605 CGTGCAGAGGTGAAGCG 552267 4-9-4 58 1316 1590 1606 ACGTGCAGAGGTGAAGC 552268 4-9-4 62 1317 1778 1794 TTATGCCTACAGCCTCC 552269 4-9-4 52 47 1779 1795 TTTATGCCTACAGCCTC 552270 4-9-4 54 49 1780 1796 ATTTATGCCTACAGCCT 552271 4-9-4 58 51 1781 1797 AATTTATGCCTACAGCC 552272 4-9-4 40 53 1782 1798 CAATTTATGCCTACAGC 552273 4-9-4 34 54 1783 1799 CCAATTTATGCCTACAG 552274 4-9-4 34 55 1784 1800 ACCAATTTATGCCTACA 552275 4-9-4 39 56

TABLE 30 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 49 224 414 429 GAGGCATAGCAGCAGG 509959 3-10-3 43 145 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 54 17 58 73 TGGAGCCACCAGCAGG 552384 2-9-5 29 1318 58 73 TGGAGCCACCAGCAGG 552440 3-9-4 58 1318 59 74 CTGGAGCCACCAGCAG 552385 2-9-5 57 1319 59 74 CTGGAGCCACCAGCAG 552441 3-9-4 42 1319 60 75 ACTGGAGCCACCAGCA 552386 2-9-5 53 1320 60 75 ACTGGAGCCACCAGCA 552442 3-9-4 53 1320 61 76 AACTGGAGCCACCAGC 552387 2-9-5 48 1321 61 76 AACTGGAGCCACCAGC 552443 3-9-4 59 1321 62 77 GAACTGGAGCCACCAG 552388 2-9-5 40 86 62 77 GAACTGGAGCCACCAG 552444 3-9-4 51 86 411 426 GCATAGCAGCAGGATG 552389 2-9-5 39 137 411 426 GCATAGCAGCAGGATG 552445 3-9-4 60 137 412 427 GGCATAGCAGCAGGAT 552390 2-9-5 52 140 412 427 GGCATAGCAGCAGGAT 552446 3-9-4 54 140 413 428 AGGCATAGCAGCAGGA 552391 2-9-5 57 143 413 428 AGGCATAGCAGCAGGA 552447 3-9-4 54 143 414 429 GAGGCATAGCAGCAGG 552392 2-9-5 0 145 414 429 GAGGCATAGCAGCAGG 552448 3-9-4 58 145 415 430 TGAGGCATAGCAGCAG 552393 2-9-5 59 147 415 430 TGAGGCATAGCAGCAG 552449 3-9-4 60 147 416 431 ATGAGGCATAGCAGCA 552394 2-9-5 53 149 416 431 ATGAGGCATAGCAGCA 552450 3-9-4 53 149 417 432 GATGAGGCATAGCAGC 552395 2-9-5 57 151 417 432 GATGAGGCATAGCAGC 552451 3-9-4 39 151 418 433 AGATGAGGCATAGCAG 552396 2-9-5 62 153 418 433 AGATGAGGCATAGCAG 552452 3-9-4 57 153 457 473 ACGGGCAACATACCTTG 552238 4-9-4 38 33 457 473 ACGGGCAACATACCTTG 552292 5-9-3 48 33 457 473 ACGGGCAACATACCTTG 552346 6-9-2 0 33 457 472 CGGGCAACATACCTTG 552397 2-9-5 63 167 457 472 CGGGCAACATACCTTG 552453 3-9-4 56 167 458 473 ACGGGCAACATACCTT 552398 2-9-5 61 168 458 473 ACGGGCAACATACCTT 552454 3-9-4 48 168 670 685 CTAGTAAACTGAGCCA 552399 2-9-5 52 181 671 686 ACTAGTAAACTGAGCC 552400 2-9-5 57 1322 672 687 CACTAGTAAACTGAGC 552401 2-9-5 52 1323 673 688 GCACTAGTAAACTGAG 552402 2-9-5 54 1324 687 702 CCACTGAACAAATGGC 552403 2-9-5 74 188 688 703 ACCACTGAACAAATGG 552404 2-9-5 43 190 689 704 AACCACTGAACAAATG 552405 2-9-5 15 191 690 705 GAACCACTGAACAAAT 552406 2-9-5 37 192 691 706 CGAACCACTGAACAAA 552407 2-9-5 37 194 1261 1276 GCAGTATGGATCGGCA 552408 2-9-5 76 211 1262 1277 CGCAGTATGGATCGGC 552409 2-9-5 76 1325 1263 1278 CCGCAGTATGGATCGG 552410 2-9-5 63 1326 1264 1279 TCCGCAGTATGGATCG 552411 2-9-5 70 1327 1265 1280 TTCCGCAGTATGGATC 552412 2-9-5 62 1328 1266 1281 GTTCCGCAGTATGGAT 552413 2-9-5 56 1329 1267 1282 AGTTCCGCAGTATGGA 552414 2-9-5 63 1330 1268 1283 GAGTTCCGCAGTATGG 552415 2-9-5 52 1331 1269 1284 GGAGTTCCGCAGTATG 552416 2-9-5 67 1332 1270 1285 AGGAGTTCCGCAGTAT 552417 2-9-5 50 1333 1577 1592 AGCGAAGTGCACACGG 552418 2-9-5 79 1334 1578 1593 AAGCGAAGTGCACACG 552419 2-9-5 70 1335 1579 1594 GAAGCGAAGTGCACAC 552420 2-9-5 71 1336 1580 1595 TGAAGCGAAGTGCACA 552421 2-9-5 69 1337 1581 1596 GTGAAGCGAAGTGCAC 552422 2-9-5 68 1338 1582 1597 GGTGAAGCGAAGTGCA 552423 2-9-5 65 1339 1583 1598 AGGTGAAGCGAAGTGC 552424 2-9-5 70 1340 1584 1599 GAGGTGAAGCGAAGTG 552425 2-9-5 51 1341 1585 1600 AGAGGTGAAGCGAAGT 552426 2-9-5 40 1342 1586 1601 CAGAGGTGAAGCGAAG 552427 2-9-5 35 1343 1587 1602 GCAGAGGTGAAGCGAA 552428 2-9-5 58 1344 1588 1603 TGCAGAGGTGAAGCGA 552429 2-9-5 46 1345 1589 1604 GTGCAGAGGTGAAGCG 552430 2-9-5 53 1346 1590 1605 CGTGCAGAGGTGAAGC 552431 2-9-5 51 1347 1591 1606 ACGTGCAGAGGTGAAG 552432 2-9-5 57 1348 1778 1793 TATGCCTACAGCCTCC 552433 2-9-5 54 230 1779 1794 TTATGCCTACAGCCTC 552434 2-9-5 44 231 1780 1795 TTTATGCCTACAGCCT 552435 2-9-5 46 232 1781 1796 ATTTATGCCTACAGCC 552436 2-9-5 36 233 1782 1797 AATTTATGCCTACAGC 552437 2-9-5 27 234 1783 1798 CAATTTATGCCTACAG 552438 2-9-5 27 235 1784 1799 CCAATTTATGCCTACA 552439 2-9-5 13 236

TABLE 31 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 35 224 414 429 GAGGCATAGCAGCAGG 509959 3-10-3 52 145 58 73 TGGAGCCACCAGCAGG 552496 4-9-3 47 1318 59 74 CTGGAGCCACCAGCAG 552497 4-9-3 57 1319 60 75 ACTGGAGCCACCAGCA 552498 4-9-3 45 1320 61 76 AACTGGAGCCACCAGC 552499 4-9-3 52 1321 62 77 GAACTGGAGCCACCAG 552500 4-9-3 46 86 411 426 GCATAGCAGCAGGATG 552501 4-9-3 44 137 412 427 GGCATAGCAGCAGGAT 552502 4-9-3 57 140 413 428 AGGCATAGCAGCAGGA 552503 4-9-3 52 143 414 429 GAGGCATAGCAGCAGG 552504 4-9-3 45 145 415 430 TGAGGCATAGCAGCAG 552505 4-9-3 56 147 416 431 ATGAGGCATAGCAGCA 552506 4-9-3 54 149 417 432 GATGAGGCATAGCAGC 552507 4-9-3 34 151 418 433 AGATGAGGCATAGCAG 552508 4-9-3 34 153 457 472 CGGGCAACATACCTTG 552509 4-9-3 48 167 458 473 ACGGGCAACATACCTT 552510 4-9-3 50 168 670 685 CTAGTAAACTGAGCCA 552455 3-9-4 66 181 670 685 CTAGTAAACTGAGCCA 552511 4-9-3 66 181 671 686 ACTAGTAAACTGAGCC 552456 3-9-4 64 1322 671 686 ACTAGTAAACTGAGCC 552512 4-9-3 62 1322 672 687 CACTAGTAAACTGAGC 552457 3-9-4 14 1323 672 687 CACTAGTAAACTGAGC 552513 4-9-3 56 1323 673 688 GCACTAGTAAACTGAG 552458 3-9-4 59 1324 673 688 GCACTAGTAAACTGAG 552514 4-9-3 52 1324 687 702 CCACTGAACAAATGGC 552459 3-9-4 69 188 687 702 CCACTGAACAAATGGC 552515 4-9-3 57 188 688 703 ACCACTGAACAAATGG 552460 3-9-4 0 190 688 703 ACCACTGAACAAATGG 552516 4-9-3 54 190 689 704 AACCACTGAACAAATG 552461 3-9-4 20 191 689 704 AACCACTGAACAAATG 552517 4-9-3 52 191 690 705 GAACCACTGAACAAAT 552462 3-9-4 46 192 690 705 GAACCACTGAACAAAT 552518 4-9-3 34 192 691 706 CGAACCACTGAACAAA 552463 3-9-4 48 194 691 706 CGAACCACTGAACAAA 552519 4-9-3 44 194 1261 1276 GCAGTATGGATCGGCA 552464 3-9-4 81 211 1261 1276 GCAGTATGGATCGGCA 552520 4-9-3 69 211 1262 1277 CGCAGTATGGATCGGC 552465 3-9-4 84 1325 1262 1277 CGCAGTATGGATCGGC 552521 4-9-3 80 1325 1263 1278 CCGCAGTATGGATCGG 552466 3-9-4 75 1326 1263 1278 CCGCAGTATGGATCGG 552522 4-9-3 76 1326 1264 1279 TCCGCAGTATGGATCG 552467 3-9-4 65 1327 1264 1279 TCCGCAGTATGGATCG 552523 4-9-3 71 1327 1265 1280 TTCCGCAGTATGGATC 552468 3-9-4 53 1328 1265 1280 TTCCGCAGTATGGATC 552524 4-9-3 43 1328 1266 1281 GTTCCGCAGTATGGAT 552469 3-9-4 51 1329 1266 1281 GTTCCGCAGTATGGAT 552525 4-9-3 57 1329 1267 1282 AGTTCCGCAGTATGGA 552470 3-9-4 46 1330 1267 1282 AGTTCCGCAGTATGGA 552526 4-9-3 60 1330 1268 1283 GAGTTCCGCAGTATGG 552471 3-9-4 54 1331 1268 1283 GAGTTCCGCAGTATGG 552527 4-9-3 72 1331 1269 1284 GGAGTTCCGCAGTATG 552472 3-9-4 78 1332 1269 1284 GGAGTTCCGCAGTATG 552528 4-9-3 78 1332 1270 1285 AGGAGTTCCGCAGTAT 552473 3-9-4 67 1333 1270 1285 AGGAGTTCCGCAGTAT 552529 4-9-3 77 1333 1577 1592 AGCGAAGTGCACACGG 552474 3-9-4 79 1334 1577 1592 AGCGAAGTGCACACGG 552530 4-9-3 78 1334 1578 1593 AAGCGAAGTGCACACG 552475 3-9-4 74 1335 1578 1593 AAGCGAAGTGCACACG 552531 4-9-3 68 1335 1579 1594 GAAGCGAAGTGCACAC 552476 3-9-4 52 1336 1580 1595 TGAAGCGAAGTGCACA 552477 3-9-4 76 1337 1581 1596 GTGAAGCGAAGTGCAC 552478 3-9-4 70 1338 1582 1597 GGTGAAGCGAAGTGCA 552479 3-9-4 67 1339 1583 1598 AGGTGAAGCGAAGTGC 552480 3-9-4 68 1340 1584 1599 GAGGTGAAGCGAAGTG 552481 3-9-4 57 1341 1585 1600 AGAGGTGAAGCGAAGT 552482 3-9-4 51 1342 1586 1601 CAGAGGTGAAGCGAAG 552483 3-9-4 48 1343 1587 1602 GCAGAGGTGAAGCGAA 552484 3-9-4 58 1344 1588 1603 TGCAGAGGTGAAGCGA 552485 3-9-4 51 1345 1589 1604 GTGCAGAGGTGAAGCG 552486 3-9-4 55 1346 1590 1605 CGTGCAGAGGTGAAGC 552487 3-9-4 62 1347 1591 1606 ACGTGCAGAGGTGAAG 552488 3-9-4 51 1348 1778 1793 TATGCCTACAGCCTCC 552489 3-9-4 49 230 1779 1794 TTATGCCTACAGCCTC 552490 3-9-4 51 231 1780 1795 TTTATGCCTACAGCCT 552491 3-9-4 51 232 1781 1796 ATTTATGCCTACAGCC 552492 3-9-4 38 233 1782 1797 AATTTATGCCTACAGC 552493 3-9-4 52 234 1783 1798 CAATTTATGCCTACAG 552494 3-9-4 17 235 1784 1799 CCAATTTATGCCTACA 552495 3-9-4 49 236

TABLE 32 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 43 224 52 414 429 GAGGCATAGCAGCAGG 509959 3-10-3 38 145 58 73 TGGAGCCACCAGCAGG 552552 5-9-2 33 1318 59 74 CTGGAGCCACCAGCAG 552553 5-9-2 46 1319 60 75 ACTGGAGCCACCAGCA 552554 5-9-2 54 1320 61 76 AACTGGAGCCACCAGC 552555 5-9-2 50 1321 62 77 GAACTGGAGCCACCAG 552556 5-9-2 46 86 411 426 GCATAGCAGCAGGATG 552557 5-9-2 57 137 412 427 GGCATAGCAGCAGGAT 552558 5-9-2 55 140 413 428 AGGCATAGCAGCAGGA 552559 5-9-2 66 143 414 429 GAGGCATAGCAGCAGG 552560 5-9-2 44 145 415 430 TGAGGCATAGCAGCAG 552561 5-9-2 48 147 416 431 ATGAGGCATAGCAGCA 552562 5-9-2 52 149 417 432 GATGAGGCATAGCAGC 552563 5-9-2 45 151 418 433 AGATGAGGCATAGCAG 552564 5-9-2 41 153 457 472 CGGGCAACATACCTTG 552565 5-9-2 54 167 458 473 ACGGGCAACATACCTT 552566 5-9-2 56 168 670 685 CTAGTAAACTGAGCCA 552567 5-9-2 71 181 671 686 ACTAGTAAACTGAGCC 552568 5-9-2 64 1322 672 687 CACTAGTAAACTGAGC 552569 5-9-2 59 1323 673 688 GCACTAGTAAACTGAG 552570 5-9-2 60 1324 687 702 CCACTGAACAAATGGC 552571 5-9-2 55 188 688 703 ACCACTGAACAAATGG 552572 5-9-2 60 190 689 704 AACCACTGAACAAATG 552573 5-9-2 24 191 690 705 GAACCACTGAACAAAT 552574 5-9-2 34 192 691 706 CGAACCACTGAACAAA 552575 5-9-2 36 194 1261 1276 GCAGTATGGATCGGCA 552576 5-9-2 67 211 1262 1277 CGCAGTATGGATCGGC 552577 5-9-2 64 1325 1263 1278 CCGCAGTATGGATCGG 552578 5-9-2 75 1326 1264 1279 TCCGCAGTATGGATCG 552579 5-9-2 75 1327 1265 1280 TTCCGCAGTATGGATC 552580 5-9-2 59 1328 1266 1281 GTTCCGCAGTATGGAT 552581 5-9-2 54 1329 1267 1282 AGTTCCGCAGTATGGA 552582 5-9-2 61 1330 1268 1283 GAGTTCCGCAGTATGG 552583 5-9-2 69 1331 1269 1284 GGAGTTCCGCAGTATG 552584 5-9-2 74 1332 1270 1285 AGGAGTTCCGCAGTAT 552585 5-9-2 62 1333 1577 1592 AGCGAAGTGCACACGG 552586 5-9-2 79 1334 1578 1593 AAGCGAAGTGCACACG 552587 5-9-2 71 1335 1579 1594 GAAGCGAAGTGCACAC 552532 4-9-3 48 1336 1579 1594 GAAGCGAAGTGCACAC 552588 5-9-2 70 1336 1580 1595 TGAAGCGAAGTGCACA 552533 4-9-3 43 1337 1580 1595 TGAAGCGAAGTGCACA 552589 5-9-2 59 1337 1581 1596 GTGAAGCGAAGTGCAC 552534 4-9-3 62 1338 1581 1596 GTGAAGCGAAGTGCAC 552590 5-9-2 70 1338 1582 1597 GGTGAAGCGAAGTGCA 552535 4-9-3 55 1339 1582 1597 GGTGAAGCGAAGTGCA 552591 5-9-2 51 1339 1583 1598 AGGTGAAGCGAAGTGC 552536 4-9-3 3 1340 1583 1598 AGGTGAAGCGAAGTGC 552592 5-9-2 50 1340 1584 1599 GAGGTGAAGCGAAGTG 552537 4-9-3 14 1341 1584 1599 GAGGTGAAGCGAAGTG 552593 5-9-2 46 1341 1585 1600 AGAGGTGAAGCGAAGT 552538 4-9-3 52 1342 1585 1600 AGAGGTGAAGCGAAGT 552594 5-9-2 55 1342 1586 1601 CAGAGGTGAAGCGAAG 552539 4-9-3 47 1343 1586 1601 CAGAGGTGAAGCGAAG 552595 5-9-2 60 1343 1587 1602 GCAGAGGTGAAGCGAA 552540 4-9-3 60 1344 1587 1602 GCAGAGGTGAAGCGAA 552596 5-9-2 63 1344 1588 1603 TGCAGAGGTGAAGCGA 552541 4-9-3 60 1345 1588 1603 TGCAGAGGTGAAGCGA 552597 5-9-2 61 1345 1589 1604 GTGCAGAGGTGAAGCG 552542 4-9-3 64 1346 1589 1604 GTGCAGAGGTGAAGCG 552598 5-9-2 57 1346 1590 1605 CGTGCAGAGGTGAAGC 552543 4-9-3 46 1347 1590 1605 CGTGCAGAGGTGAAGC 552600 5-9-2 59 1347 1591 1606 ACGTGCAGAGGTGAAG 552544 4-9-3 53 1348 1591 1606 ACGTGCAGAGGTGAAG 552602 5-9-2 6 1348 1778 1793 TATGCCTACAGCCTCC 552545 4-9-3 33 230 1778 1793 TATGCCTACAGCCTCC 552604 5-9-2 47 230 1779 1794 TTATGCCTACAGCCTC 552546 4-9-3 42 231 1779 1794 TTATGCCTACAGCCTC 552606 5-9-2 53 231 1780 1795 TTTATGCCTACAGCCT 552547 4-9-3 51 232 1780 1795 TTTATGCCTACAGCCT 552608 5-9-2 53 232 1781 1796 ATTTATGCCTACAGCC 552548 4-9-3 52 233 1781 1796 ATTTATGCCTACAGCC 552610 5-9-2 47 233 1782 1797 AATTTATGCCTACAGC 552549 4-9-3 38 234 1782 1797 AATTTATGCCTACAGC 552612 5-9-2 39 234 1783 1798 CAATTTATGCCTACAG 552550 4-9-3 19 235 1783 1798 CAATTTATGCCTACAG 552614 5-9-2 24 235 1784 1799 CCAATTTATGCCTACA 552551 4-9-3 24 236 1784 1799 CCAATTTATGCCTACA 552616 5-9-2 15 236

TABLE 33 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 51 224 1780 1799 CCAATTTATGCCTACAGCCT 509934 5-10-5 76 50 58 77 GAACTGGAGCCACCAGCAGG 552007 6-10-4 61 83 58 77 GAACTGGAGCCACCAGCAGG 552039 7-10-3 84 83 253 272 AGAGAAGTCCACCACGAGTC 552008 6-10-4 48 103 253 272 AGAGAAGTCCACCACGAGTC 552040 7-10-3 48 103 411 430 TGAGGCATAGCAGCAGGATG 552009 6-10-4 77 136 411 430 TGAGGCATAGCAGCAGGATG 552041 7-10-3 73 136 412 431 ATGAGGCATAGCAGCAGGAT 552010 6-10-4 63 139 412 431 ATGAGGCATAGCAGCAGGAT 552042 7-10-3 66 139 413 432 GATGAGGCATAGCAGCAGGA 552011 6-10-4 52 142 413 432 GATGAGGCATAGCAGCAGGA 552043 7-10-3 54 142 414 433 AGATGAGGCATAGCAGCAGG 552012 6-10-4 73 20 414 433 AGATGAGGCATAGCAGCAGG 552044 7-10-3 86 20 415 434 AAGATGAGGCATAGCAGCAG 552013 6-10-4 73 22 415 434 AAGATGAGGCATAGCAGCAG 552045 7-10-3 65 22 416 435 GAAGATGAGGCATAGCAGCA 552014 6-10-4 76 24 416 435 GAAGATGAGGCATAGCAGCA 552046 7-10-3 93 24 417 436 AGAAGATGAGGCATAGCAGC 552015 6-10-4 70 26 417 436 AGAAGATGAGGCATAGCAGC 552047 7-10-3 77 26 418 437 AAGAAGATGAGGCATAGCAG 552016 6-10-4 61 28 418 437 AAGAAGATGAGGCATAGCAG 552048 7-10-3 66 28 687 706 CGAACCACTGAACAAATGGC 552017 6-10-4 73 39 687 706 CGAACCACTGAACAAATGGC 552049 7-10-3 73 39 1261 1280 TTCCGCAGTATGGATCGGCA 552018 6-10-4 98 719 1261 1280 TTCCGCAGTATGGATCGGCA 552050 7-10-3 98 719 1262 1281 GTTCCGCAGTATGGATCGGC 552019 6-10-4 98 212 1262 1281 GTTCCGCAGTATGGATCGGC 552051 7-10-3 99 212 1263 1282 AGTTCCGCAGTATGGATCGG 551986 4-10-6 92 720 1263 1282 AGTTCCGCAGTATGGATCGG 552020 6-10-4 97 720 1263 1282 AGTTCCGCAGTATGGATCGG 552052 7-10-3 98 720 1264 1283 GAGTTCCGCAGTATGGATCG 551987 4-10-6 95 721 1264 1283 GAGTTCCGCAGTATGGATCG 552021 6-10-4 97 721 1264 1283 GAGTTCCGCAGTATGGATCG 552053 7-10-3 98 721 1265 1284 GGAGTTCCGCAGTATGGATC 551988 4-10-6 50 1349 1265 1284 GGAGTTCCGCAGTATGGATC 552005 5-10-5 99 1349 1265 1284 GGAGTTCCGCAGTATGGATC 552022 6-10-4 99 1349 1265 1284 GGAGTTCCGCAGTATGGATC 552054 7-10-3 99 1349 1266 1285 AGGAGTTCCGCAGTATGGAT 551989 4-10-6 96 722 1266 1285 AGGAGTTCCGCAGTATGGAT 552023 6-10-4 99 722 1266 1285 AGGAGTTCCGCAGTATGGAT 552055 7-10-3 98 722 1577 1596 GTGAAGCGAAGTGCACACGG 551990 4-10-6 86 224 1577 1596 GTGAAGCGAAGTGCACACGG 552024 6-10-4 89 224 1577 1596 GTGAAGCGAAGTGCACACGG 552056 7-10-3 88 224 1578 1597 GGTGAAGCGAAGTGCACACG 551991 4-10-6 0 801 1578 1597 GGTGAAGCGAAGTGCACACG 552025 6-10-4 90 801 1578 1597 GGTGAAGCGAAGTGCACACG 552057 7-10-3 92 801 1579 1598 AGGTGAAGCGAAGTGCACAC 551992 4-10-6 72 802 1579 1598 AGGTGAAGCGAAGTGCACAC 552026 6-10-4 88 802 1579 1598 AGGTGAAGCGAAGTGCACAC 552058 7-10-3 86 802 1580 1599 GAGGTGAAGCGAAGTGCACA 551993 4-10-6 82 225 1580 1599 GAGGTGAAGCGAAGTGCACA 552027 6-10-4 87 225 1580 1599 GAGGTGAAGCGAAGTGCACA 552059 7-10-3 88 225 1581 1600 AGAGGTGAAGCGAAGTGCAC 551994 4-10-6 85 804 1581 1600 AGAGGTGAAGCGAAGTGCAC 552028 6-10-4 83 804 1581 1600 AGAGGTGAAGCGAAGTGCAC 552060 7-10-3 82 804 1582 1601 CAGAGGTGAAGCGAAGTGCA 551995 4-10-6 84 805 1582 1601 CAGAGGTGAAGCGAAGTGCA 552029 6-10-4 88 805 1582 1601 CAGAGGTGAAGCGAAGTGCA 552061 7-10-3 85 805 1583 1602 GCAGAGGTGAAGCGAAGTGC 551996 4-10-6 87 226 1583 1602 GCAGAGGTGAAGCGAAGTGC 552030 6-10-4 88 226 1583 1602 GCAGAGGTGAAGCGAAGTGC 552062 7-10-3 85 226 1584 1603 TGCAGAGGTGAAGCGAAGTG 551997 4-10-6 83 806 1584 1603 TGCAGAGGTGAAGCGAAGTG 552031 6-10-4 82 806 1585 1604 GTGCAGAGGTGAAGCGAAGT 551998 4-10-6 85 807 1585 1604 GTGCAGAGGTGAAGCGAAGT 552032 6-10-4 87 807 1586 1605 CGTGCAGAGGTGAAGCGAAG 551999 4-10-6 82 227 1586 1605 CGTGCAGAGGTGAAGCGAAG 552033 6-10-4 87 227 1587 1606 ACGTGCAGAGGTGAAGCGAA 552000 4-10-6 83 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 552006 5-10-5 88 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 552034 6-10-4 89 1350 1778 1797 AATTTATGCCTACAGCCTCC 552001 4-10-6 65 46 1778 1797 AATTTATGCCTACAGCCTCC 552035 6-10-4 60 46 1779 1798 CAATTTATGCCTACAGCCTC 552002 4-10-6 63 48 1779 1798 CAATTTATGCCTACAGCCTC 552036 6-10-4 65 48 1780 1799 CCAATTTATGCCTACAGCCT 552003 4-10-6 65 50 1780 1799 CCAATTTATGCCTACAGCCT 552037 6-10-4 58 50 1781 1800 ACCAATTTATGCCTACAGCC 552004 4-10-6 58 52 1781 1800 ACCAATTTATGCCTACAGCC 552038 6-10-4 70 52

TABLE 34 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 64 224 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 62 17 58 74 CTGGAGCCACCAGCAGG 552168 3-9-5 79 1288 58 74 CTGGAGCCACCAGCAGG 552222 4-9-4 79 1288 59 75 ACTGGAGCCACCAGCAG 552169 3-9-5 67 1289 59 75 ACTGGAGCCACCAGCAG 552223 4-9-4 40 1289 60 76 AACTGGAGCCACCAGCA 552170 3-9-5 69 1290 60 76 AACTGGAGCCACCAGCA 552224 4-9-4 64 1290 61 77 GAACTGGAGCCACCAGC 552171 3-9-5 65 1291 61 77 GAACTGGAGCCACCAGC 552225 4-9-4 69 1291 253 269 GAAGTCCACCACGAGTC 552172 3-9-5 33 9 253 269 GAAGTCCACCACGAGTC 552226 4-9-4 48 9 254 270 AGAAGTCCACCACGAGT 552173 3-9-5 41 10 254 270 AGAAGTCCACCACGAGT 552227 4-9-4 32 10 255 271 GAGAAGTCCACCACGAG 552174 3-9-5 31 11 255 271 GAGAAGTCCACCACGAG 552228 4-9-4 42 11 256 272 AGAGAAGTCCACCACGA 552175 3-9-5 59 12 411 427 GGCATAGCAGCAGGATG 552176 3-9-5 68 17 412 428 AGGCATAGCAGCAGGAT 552177 3-9-5 55 18 413 429 GAGGCATAGCAGCAGGA 552178 3-9-5 66 19 414 430 TGAGGCATAGCAGCAGG 552179 3-9-5 70 21 415 431 ATGAGGCATAGCAGCAG 552180 3-9-5 66 23 416 432 GATGAGGCATAGCAGCA 552181 3-9-5 51 25 417 433 AGATGAGGCATAGCAGC 552182 3-9-5 69 27 418 434 AAGATGAGGCATAGCAG 552183 3-9-5 69 29 457 473 ACGGGCAACATACCTTG 552184 3-9-5 43 33 670 686 ACTAGTAAACTGAGCCA 552185 3-9-5 66 1292 671 687 CACTAGTAAACTGAGCC 552186 3-9-5 54 1293 672 688 GCACTAGTAAACTGAGC 552187 3-9-5 74 1294 687 703 ACCACTGAACAAATGGC 552188 3-9-5 78 40 688 704 AACCACTGAACAAATGG 552189 3-9-5 57 41 689 705 GAACCACTGAACAAATG 552190 3-9-5 39 42 690 706 CGAACCACTGAACAAAT 552191 3-9-5 60 43 1261 1277 CGCAGTATGGATCGGCA 552192 3-9-5 85 1295 1262 1278 CCGCAGTATGGATCGGC 552193 3-9-5 86 1296 1263 1279 TCCGCAGTATGGATCGG 552194 3-9-5 68 1297 1264 1280 TTCCGCAGTATGGATCG 552195 3-9-5 73 1298 1265 1281 GTTCCGCAGTATGGATC 552196 3-9-5 60 1299 1266 1282 AGTTCCGCAGTATGGAT 552197 3-9-5 60 1300 1267 1283 GAGTTCCGCAGTATGGA 552198 3-9-5 61 1301 1268 1284 GGAGTTCCGCAGTATGG 552199 3-9-5 89 1302 1269 1285 AGGAGTTCCGCAGTATG 552200 3-9-5 85 1303 1577 1593 AAGCGAAGTGCACACGG 552201 3-9-5 81 1304 1578 1594 GAAGCGAAGTGCACACG 552202 3-9-5 76 1305 1579 1595 TGAAGCGAAGTGCACAC 552203 3-9-5 74 1306 1580 1596 GTGAAGCGAAGTGCACA 552204 3-9-5 71 1307 1581 1597 GGTGAAGCGAAGTGCAC 552151 2-9-6 77 1308 1581 1597 GGTGAAGCGAAGTGCAC 552205 3-9-5 78 1308 1582 1598 AGGTGAAGCGAAGTGCA 552152 2-9-6 72 1309 1582 1598 AGGTGAAGCGAAGTGCA 552206 3-9-5 77 1309 1583 1599 GAGGTGAAGCGAAGTGC 552153 2-9-6 67 1310 1583 1599 GAGGTGAAGCGAAGTGC 552207 3-9-5 81 1310 1584 1600 AGAGGTGAAGCGAAGTG 552154 2-9-6 56 1311 1584 1600 AGAGGTGAAGCGAAGTG 552208 3-9-5 70 1311 1585 1601 CAGAGGTGAAGCGAAGT 552155 2-9-6 61 1312 1585 1601 CAGAGGTGAAGCGAAGT 552209 3-9-5 63 1312 1586 1602 GCAGAGGTGAAGCGAAG 552156 2-9-6 20 1313 1586 1602 GCAGAGGTGAAGCGAAG 552210 3-9-5 75 1313 1587 1603 TGCAGAGGTGAAGCGAA 552157 2-9-6 39 1314 1587 1603 TGCAGAGGTGAAGCGAA 552211 3-9-5 75 1314 1588 1604 GTGCAGAGGTGAAGCGA 552158 2-9-6 70 1315 1588 1604 GTGCAGAGGTGAAGCGA 552212 3-9-5 67 1315 1589 1605 CGTGCAGAGGTGAAGCG 552159 2-9-6 74 1316 1589 1605 CGTGCAGAGGTGAAGCG 552213 3-9-5 70 1316 1590 1606 ACGTGCAGAGGTGAAGC 552160 2-9-6 78 1317 1590 1606 ACGTGCAGAGGTGAAGC 552214 3-9-5 79 1317 1778 1794 TTATGCCTACAGCCTCC 552161 2-9-6 56 47 1778 1794 TTATGCCTACAGCCTCC 552215 3-9-5 61 47 1779 1795 TTTATGCCTACAGCCTC 552162 2-9-6 64 49 1779 1795 TTTATGCCTACAGCCTC 552216 3-9-5 62 49 1780 1796 ATTTATGCCTACAGCCT 552163 2-9-6 71 51 1780 1796 ATTTATGCCTACAGCCT 552217 3-9-5 58 51 1781 1797 AATTTATGCCTACAGCC 552164 2-9-6 52 53 1781 1797 AATTTATGCCTACAGCC 552218 3-9-5 56 53 1782 1798 CAATTTATGCCTACAGC 552165 2-9-6 53 54 1782 1798 CAATTTATGCCTACAGC 552219 3-9-5 33 54 1783 1799 CCAATTTATGCCTACAG 552166 2-9-6 41 55 1783 1799 CCAATTTATGCCTACAG 552220 3-9-5 53 55 1784 1800 ACCAATTTATGCCTACA 552167 2-9-6 54 56 1784 1800 ACCAATTTATGCCTACA 552221 3-9-5 31 56

TABLE 35 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 60 224 85 1780 1799 CCAATTTATGCCTACAGCCT 509934 5-10-5 76 50 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 73 17 58 77 GAACTGGAGCCACCAGCAGG 552071 8-10-2 79 83 58 74 CTGGAGCCACCAGCAGG 552114 2-9-6 66 1288 59 75 ACTGGAGCCACCAGCAG 552115 2-9-6 70 1289 60 76 AACTGGAGCCACCAGCA 552116 2-9-6 68 1290 61 77 GAACTGGAGCCACCAGC 552117 2-9-6 70 1291 253 272 AGAGAAGTCCACCACGAGTC 552072 8-10-2 50 103 253 269 GAAGTCCACCACGAGTC 552118 2-9-6 66 9 254 270 AGAAGTCCACCACGAGT 552119 2-9-6 62 10 255 271 GAGAAGTCCACCACGAG 552120 2-9-6 35 11 256 272 AGAGAAGTCCACCACGA 552121 2-9-6 39 12 411 430 TGAGGCATAGCAGCAGGATG 552073 8-10-2 80 136 411 427 GGCATAGCAGCAGGATG 552122 2-9-6 55 17 412 431 ATGAGGCATAGCAGCAGGAT 552074 8-10-2 73 139 412 428 AGGCATAGCAGCAGGAT 552123 2-9-6 75 18 413 432 GATGAGGCATAGCAGCAGGA 552075 8-10-2 78 142 413 429 GAGGCATAGCAGCAGGA 552124 2-9-6 64 19 414 433 AGATGAGGCATAGCAGCAGG 552076 8-10-2 70 20 414 430 TGAGGCATAGCAGCAGG 552125 2-9-6 73 21 415 434 AAGATGAGGCATAGCAGCAG 552077 8-10-2 83 22 415 431 ATGAGGCATAGCAGCAG 552126 2-9-6 64 23 416 435 GAAGATGAGGCATAGCAGCA 552078 8-10-2 80 24 416 432 GATGAGGCATAGCAGCA 552127 2-9-6 72 25 417 436 AGAAGATGAGGCATAGCAGC 552079 8-10-2 86 26 417 433 AGATGAGGCATAGCAGC 552128 2-9-6 76 27 418 437 AAGAAGATGAGGCATAGCAG 552080 8-10-2 83 28 418 434 AAGATGAGGCATAGCAG 552129 2-9-6 72 29 670 686 ACTAGTAAACTGAGCCA 552131 2-9-6 61 1292 671 687 CACTAGTAAACTGAGCC 552132 2-9-6 73 1293 672 688 GCACTAGTAAACTGAGC 552133 2-9-6 75 1294 687 706 CGAACCACTGAACAAATGGC 552081 8-10-2 76 39 687 703 ACCACTGAACAAATGGC 552134 2-9-6 58 40 688 704 AACCACTGAACAAATGG 552135 2-9-6 67 41 689 705 GAACCACTGAACAAATG 552136 2-9-6 65 42 690 706 CGAACCACTGAACAAAT 552137 2-9-6 55 43 1261 1280 TTCCGCAGTATGGATCGGCA 552082 8-10-2 98 719 1261 1277 CGCAGTATGGATCGGCA 552138 2-9-6 82 1295 1262 1281 GTTCCGCAGTATGGATCGGC 552083 8-10-2 99 212 1262 1278 CCGCAGTATGGATCGGC 552139 2-9-6 86 1296 1263 1282 AGTTCCGCAGTATGGATCGG 552084 8-10-2 99 720 1263 1279 TCCGCAGTATGGATCGG 552140 2-9-6 74 1297 1264 1283 GAGTTCCGCAGTATGGATCG 552085 8-10-2 100 721 1264 1280 TTCCGCAGTATGGATCG 552141 2-9-6 67 1298 1265 1284 GGAGTTCCGCAGTATGGATC 552086 8-10-2 100 1349 1265 1281 GTTCCGCAGTATGGATC 552142 2-9-6 45 1299 1266 1285 AGGAGTTCCGCAGTATGGAT 552087 8-10-2 100 722 1266 1282 AGTTCCGCAGTATGGAT 552143 2-9-6 68 1300 1267 1283 GAGTTCCGCAGTATGGA 552144 2-9-6 78 1301 1268 1284 GGAGTTCCGCAGTATGG 552145 2-9-6 88 1302 1269 1285 AGGAGTTCCGCAGTATG 552146 2-9-6 81 1303 1577 1596 GTGAAGCGAAGTGCACACGG 552088 8-10-2 95 224 1577 1593 AAGCGAAGTGCACACGG 552147 2-9-6 88 1304 1578 1597 GGTGAAGCGAAGTGCACACG 552089 8-10-2 93 801 1578 1594 GAAGCGAAGTGCACACG 552148 2-9-6 79 1305 1579 1598 AGGTGAAGCGAAGTGCACAC 552090 8-10-2 87 802 1579 1595 TGAAGCGAAGTGCACAC 552149 2-9-6 81 1306 1580 1599 GAGGTGAAGCGAAGTGCACA 552091 8-10-2 88 225 1581 1600 AGAGGTGAAGCGAAGTGCAC 552092 8-10-2 90 804 1582 1601 CAGAGGTGAAGCGAAGTGCA 552093 8-10-2 91 805 1583 1602 GCAGAGGTGAAGCGAAGTGC 552094 8-10-2 88 226 1584 1603 TGCAGAGGTGAAGCGAAGTG 552063 7-10-3 81 806 1584 1603 TGCAGAGGTGAAGCGAAGTG 552095 8-10-2 89 806 1585 1604 GTGCAGAGGTGAAGCGAAGT 552064 7-10-3 85 807 1585 1604 GTGCAGAGGTGAAGCGAAGT 552096 8-10-2 92 807 1586 1605 CGTGCAGAGGTGAAGCGAAG 552065 7-10-3 86 227 1586 1605 CGTGCAGAGGTGAAGCGAAG 552097 8-10-2 93 227 1587 1606 ACGTGCAGAGGTGAAGCGAA 552066 7-10-3 33 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 552098 8-10-2 88 1350 1778 1797 AATTTATGCCTACAGCCTCC 552067 7-10-3 50 46 1778 1797 AATTTATGCCTACAGCCTCC 552099 8-10-2 70 46 1779 1798 CAATTTATGCCTACAGCCTC 552068 7-10-3 73 48 1779 1798 CAATTTATGCCTACAGCCTC 552100 8-10-2 70 48 1780 1799 CCAATTTATGCCTACAGCCT 552069 7-10-3 73 50 1780 1799 CCAATTTATGCCTACAGCCT 552101 8-10-2 76 50 1781 1800 ACCAATTTATGCCTACAGCC 552070 7-10-3 71 52 1781 1800 ACCAATTTATGCCTACAGCC 552102 8-10-2 64 52

TABLE 36 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 84 224 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 76 17 58 74 CTGGAGCCACCAGCAGG 552330 6-9-2 54 1288 59 75 ACTGGAGCCACCAGCAG 552331 6-9-2 66 1289 60 76 AACTGGAGCCACCAGCA 552332 6-9-2 70 1290 61 77 GAACTGGAGCCACCAGC 552333 6-9-2 55 1291 253 269 GAAGTCCACCACGAGTC 552334 6-9-2 42 9 254 270 AGAAGTCCACCACGAGT 552335 6-9-2 39 10 255 271 GAGAAGTCCACCACGAG 552336 6-9-2 27 11 256 272 AGAGAAGTCCACCACGA 552337 6-9-2 74 12 411 427 GGCATAGCAGCAGGATG 552338 6-9-2 68 17 412 428 AGGCATAGCAGCAGGAT 552339 6-9-2 71 18 413 429 GAGGCATAGCAGCAGGA 552340 6-9-2 61 19 414 430 TGAGGCATAGCAGCAGG 552341 6-9-2 58 21 415 431 ATGAGGCATAGCAGCAG 552342 6-9-2 55 23 416 432 GATGAGGCATAGCAGCA 552343 6-9-2 63 25 417 433 AGATGAGGCATAGCAGC 552344 6-9-2 51 27 418 434 AAGATGAGGCATAGCAG 552345 6-9-2 65 29 457 473 ACGGGCAACATACCTTG 552346 6-9-2 0 33 670 686 ACTAGTAAACTGAGCCA 552347 6-9-2 84 1292 671 687 CACTAGTAAACTGAGCC 552348 6-9-2 87 1293 672 688 GCACTAGTAAACTGAGC 552349 6-9-2 74 1294 687 703 ACCACTGAACAAATGGC 552350 6-9-2 59 40 688 704 AACCACTGAACAAATGG 552351 6-9-2 60 41 689 705 GAACCACTGAACAAATG 552352 6-9-2 53 42 690 706 CGAACCACTGAACAAAT 552353 6-9-2 0 43 1261 1277 CGCAGTATGGATCGGCA 552354 6-9-2 83 1295 1262 1278 CCGCAGTATGGATCGGC 552355 6-9-2 90 1296 1263 1279 TCCGCAGTATGGATCGG 552356 6-9-2 0 1297 1264 1280 TTCCGCAGTATGGATCG 552357 6-9-2 45 1298 1265 1281 GTTCCGCAGTATGGATC 552358 6-9-2 74 1299 1266 1282 AGTTCCGCAGTATGGAT 552359 6-9-2 72 1300 1267 1283 GAGTTCCGCAGTATGGA 552360 6-9-2 87 1301 1268 1284 GGAGTTCCGCAGTATGG 552361 6-9-2 96 1302 1269 1285 AGGAGTTCCGCAGTATG 552308 5-9-3 81 1303 1269 1285 AGGAGTTCCGCAGTATG 552362 6-9-2 92 1303 1577 1593 AAGCGAAGTGCACACGG 552309 5-9-3 77 1304 1577 1593 AAGCGAAGTGCACACGG 552363 6-9-2 92 1304 1578 1594 GAAGCGAAGTGCACACG 552310 5-9-3 80 1305 1578 1594 GAAGCGAAGTGCACACG 552364 6-9-2 87 1305 1579 1595 TGAAGCGAAGTGCACAC 552311 5-9-3 13 1306 1579 1595 TGAAGCGAAGTGCACAC 552365 6-9-2 84 1306 1580 1596 GTGAAGCGAAGTGCACA 552150 2-9-6 73 1307 1580 1596 GTGAAGCGAAGTGCACA 552312 5-9-3 77 1307 1580 1596 GTGAAGCGAAGTGCACA 552366 6-9-2 87 1307 1581 1597 GGTGAAGCGAAGTGCAC 552313 5-9-3 64 1308 1581 1597 GGTGAAGCGAAGTGCAC 552367 6-9-2 85 1308 1582 1598 AGGTGAAGCGAAGTGCA 552314 5-9-3 73 1309 1582 1598 AGGTGAAGCGAAGTGCA 552368 6-9-2 77 1309 1583 1599 GAGGTGAAGCGAAGTGC 552315 5-9-3 75 1310 1583 1599 GAGGTGAAGCGAAGTGC 552369 6-9-2 75 1310 1584 1600 AGAGGTGAAGCGAAGTG 552316 5-9-3 64 1311 1584 1600 AGAGGTGAAGCGAAGTG 552370 6-9-2 63 1311 1585 1601 CAGAGGTGAAGCGAAGT 552317 5-9-3 99 1312 1585 1601 CAGAGGTGAAGCGAAGT 552371 6-9-2 81 1312 1586 1602 GCAGAGGTGAAGCGAAG 552318 5-9-3 76 1313 1586 1602 GCAGAGGTGAAGCGAAG 552372 6-9-2 65 1313 1587 1603 TGCAGAGGTGAAGCGAA 552319 5-9-3 55 1314 1587 1603 TGCAGAGGTGAAGCGAA 552373 6-9-2 74 1314 1588 1604 GTGCAGAGGTGAAGCGA 552320 5-9-3 68 1315 1588 1604 GTGCAGAGGTGAAGCGA 552374 6-9-2 78 1315 1589 1605 CGTGCAGAGGTGAAGCG 552321 5-9-3 74 1316 1589 1605 CGTGCAGAGGTGAAGCG 552375 6-9-2 81 1316 1590 1606 ACGTGCAGAGGTGAAGC 552322 5-9-3 73 1317 1590 1606 ACGTGCAGAGGTGAAGC 552376 6-9-2 78 1317 1778 1794 TTATGCCTACAGCCTCC 552323 5-9-3 75 47 1778 1794 TTATGCCTACAGCCTCC 552377 6-9-2 70 47 1779 1795 TTTATGCCTACAGCCTC 552324 5-9-3 0 49 1779 1795 TTTATGCCTACAGCCTC 552378 6-9-2 72 49 1780 1796 ATTTATGCCTACAGCCT 552325 5-9-3 70 51 1780 1796 ATTTATGCCTACAGCCT 552379 6-9-2 74 51 1781 1797 AATTTATGCCTACAGCC 552326 5-9-3 63 53 1781 1797 AATTTATGCCTACAGCC 552380 6-9-2 53 53 1782 1798 CAATTTATGCCTACAGC 552327 5-9-3 30 54 1782 1798 CAATTTATGCCTACAGC 552381 6-9-2 26 54 1783 1799 CCAATTTATGCCTACAG 552328 5-9-3 25 55 1783 1799 CCAATTTATGCCTACAG 552382 6-9-2 13 55 1784 1800 ACCAATTTATGCCTACA 552329 5-9-3 33 56 1784 1800 ACCAATTTATGCCTACA 552383 6-9-2 5 56

TABLE 37 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1780 1799 CCAATTTATGCCTACAGCCT 509934 5-10-5 30 50 58 77 GAACTGGAGCCACCAGCAGG 551909 2-10-8 62 83 58 77 GAACTGGAGCCACCAGCAGG 551941 3-10-7 74 83 58 77 GAACTGGAGCCACCAGCAGG 551973 4-10-6 64 83 253 272 AGAGAAGTCCACCACGAGTC 551910 2-10-8 52 103 253 272 AGAGAAGTCCACCACGAGTC 551942 3-10-7 54 103 253 272 AGAGAAGTCCACCACGAGTC 551974 4-10-6 51 103 411 430 TGAGGCATAGCAGCAGGATG 551911 2-10-8 58 136 411 430 TGAGGCATAGCAGCAGGATG 551943 3-10-7 64 136 411 430 TGAGGCATAGCAGCAGGATG 551975 4-10-6 57 136 412 431 ATGAGGCATAGCAGCAGGAT 551912 2-10-8 59 139 412 431 ATGAGGCATAGCAGCAGGAT 551944 3-10-7 66 139 412 431 ATGAGGCATAGCAGCAGGAT 551976 4-10-6 57 139 413 432 GATGAGGCATAGCAGCAGGA 551913 2-10-8 58 142 413 432 GATGAGGCATAGCAGCAGGA 551945 3-10-7 56 142 413 432 GATGAGGCATAGCAGCAGGA 551977 4-10-6 56 142 414 433 AGATGAGGCATAGCAGCAGG 551914 2-10-8 0 20 414 433 AGATGAGGCATAGCAGCAGG 551946 3-10-7 48 20 414 433 AGATGAGGCATAGCAGCAGG 551978 4-10-6 53 20 415 434 AAGATGAGGCATAGCAGCAG 551915 2-10-8 44 22 415 434 AAGATGAGGCATAGCAGCAG 551947 3-10-7 53 22 415 434 AAGATGAGGCATAGCAGCAG 551979 4-10-6 64 22 416 435 GAAGATGAGGCATAGCAGCA 551916 2-10-8 57 24 416 435 GAAGATGAGGCATAGCAGCA 551948 3-10-7 68 24 416 435 GAAGATGAGGCATAGCAGCA 551980 4-10-6 56 24 417 436 AGAAGATGAGGCATAGCAGC 551917 2-10-8 58 26 417 436 AGAAGATGAGGCATAGCAGC 551949 3-10-7 64 26 417 436 AGAAGATGAGGCATAGCAGC 551981 4-10-6 63 26 418 437 AAGAAGATGAGGCATAGCAG 551918 2-10-8 59 28 418 437 AAGAAGATGAGGCATAGCAG 551950 3-10-7 71 28 418 437 AAGAAGATGAGGCATAGCAG 551982 4-10-6 63 28 687 706 CGAACCACTGAACAAATGGC 551919 2-10-8 76 39 687 706 CGAACCACTGAACAAATGGC 551951 3-10-7 71 39 687 706 CGAACCACTGAACAAATGGC 551983 4-10-6 73 39 1261 1280 TTCCGCAGTATGGATCGGCA 551920 2-10-8 68 719 1261 1280 TTCCGCAGTATGGATCGGCA 551952 3-10-7 76 719 1261 1280 TTCCGCAGTATGGATCGGCA 551984 4-10-6 81 719 1262 1281 GTTCCGCAGTATGGATCGGC 551921 2-10-8 83 212 1262 1281 GTTCCGCAGTATGGATCGGC 551953 3-10-7 82 212 1262 1281 GTTCCGCAGTATGGATCGGC 551985 4-10-6 76 212 1263 1282 AGTTCCGCAGTATGGATCGG 551922 2-10-8 73 720 1263 1282 AGTTCCGCAGTATGGATCGG 551954 3-10-7 68 720 1264 1283 GAGTTCCGCAGTATGGATCG 551923 2-10-8 59 721 1264 1283 GAGTTCCGCAGTATGGATCG 551955 3-10-7 71 721 1265 1284 GGAGTTCCGCAGTATGGATC 551924 2-10-8 80 1349 1265 1284 GGAGTTCCGCAGTATGGATC 551956 3-10-7 80 1349 1266 1285 AGGAGTTCCGCAGTATGGAT 551925 2-10-8 82 722 1266 1285 AGGAGTTCCGCAGTATGGAT 551957 3-10-7 88 722 1577 1596 GTGAAGCGAAGTGCACACGG 551926 2-10-8 71 224 1577 1596 GTGAAGCGAAGTGCACACGG 551958 3-10-7 74 224 1578 1597 GGTGAAGCGAAGTGCACACG 551927 2-10-8 68 801 1578 1597 GGTGAAGCGAAGTGCACACG 551959 3-10-7 69 801 1579 1598 AGGTGAAGCGAAGTGCACAC 551928 2-10-8 69 802 1579 1598 AGGTGAAGCGAAGTGCACAC 551960 3-10-7 62 802 1580 1599 GAGGTGAAGCGAAGTGCACA 551929 2-10-8 54 225 1580 1599 GAGGTGAAGCGAAGTGCACA 551961 3-10-7 20 225 1581 1600 AGAGGTGAAGCGAAGTGCAC 551930 2-10-8 53 804 1581 1600 AGAGGTGAAGCGAAGTGCAC 551962 3-10-7 60 804 1582 1601 CAGAGGTGAAGCGAAGTGCA 551931 2-10-8 47 805 1582 1601 CAGAGGTGAAGCGAAGTGCA 551963 3-10-7 63 805 1583 1602 GCAGAGGTGAAGCGAAGTGC 551932 2-10-8 68 226 1583 1602 GCAGAGGTGAAGCGAAGTGC 551964 3-10-7 56 226 1584 1603 TGCAGAGGTGAAGCGAAGTG 551933 2-10-8 72 806 1584 1603 TGCAGAGGTGAAGCGAAGTG 551965 3-10-7 67 806 1585 1604 GTGCAGAGGTGAAGCGAAGT 551934 2-10-8 64 807 1585 1604 GTGCAGAGGTGAAGCGAAGT 551966 3-10-7 73 807 1586 1605 CGTGCAGAGGTGAAGCGAAG 551935 2-10-8 68 227 1586 1605 CGTGCAGAGGTGAAGCGAAG 551967 3-10-7 60 227 1587 1606 ACGTGCAGAGGTGAAGCGAA 551936 2-10-8 67 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 551968 3-10-7 63 1350 1778 1797 AATTTATGCCTACAGCCTCC 551937 2-10-8 47 46 1778 1797 AATTTATGCCTACAGCCTCC 551969 3-10-7 36 46 1779 1798 CAATTTATGCCTACAGCCTC 551938 2-10-8 41 48 1779 1798 CAATTTATGCCTACAGCCTC 551970 3-10-7 43 48 1780 1799 CCAATTTATGCCTACAGCCT 551939 2-10-8 53 50 1780 1799 CCAATTTATGCCTACAGCCT 551971 3-10-7 55 50 1781 1800 ACCAATTTATGCCTACAGCC 551940 2-10-8 50 52 1781 1800 ACCAATTTATGCCTACAGCC 551972 3-10-7 58 52

TABLE 38 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1780 1799 CCAATTTATGCCTACAGCCT 509934 5-10-5 21 50 58 77 GAACTGGAGCCACCAGCAGG 551909 2-10-8 52 83 58 77 GAACTGGAGCCACCAGCAGG 551941 3-10-7 62 83 58 77 GAACTGGAGCCACCAGCAGG 551973 4-10-6 58 83 253 272 AGAGAAGTCCACCACGAGTC 551910 2-10-8 48 103 253 272 AGAGAAGTCCACCACGAGTC 551942 3-10-7 36 103 253 272 AGAGAAGTCCACCACGAGTC 551974 4-10-6 45 103 411 430 TGAGGCATAGCAGCAGGATG 551911 2-10-8 61 136 411 430 TGAGGCATAGCAGCAGGATG 551943 3-10-7 56 136 411 430 TGAGGCATAGCAGCAGGATG 551975 4-10-6 60 136 412 431 ATGAGGCATAGCAGCAGGAT 551912 2-10-8 53 139 412 431 ATGAGGCATAGCAGCAGGAT 551944 3-10-7 48 139 412 431 ATGAGGCATAGCAGCAGGAT 551976 4-10-6 48 139 413 432 GATGAGGCATAGCAGCAGGA 551913 2-10-8 53 142 413 432 GATGAGGCATAGCAGCAGGA 551945 3-10-7 54 142 413 432 GATGAGGCATAGCAGCAGGA 551977 4-10-6 48 142 414 433 AGATGAGGCATAGCAGCAGG 551914 2-10-8 0 20 414 433 AGATGAGGCATAGCAGCAGG 551946 3-10-7 56 20 414 433 AGATGAGGCATAGCAGCAGG 551978 4-10-6 36 20 415 434 AAGATGAGGCATAGCAGCAG 551915 2-10-8 47 22 415 434 AAGATGAGGCATAGCAGCAG 551947 3-10-7 45 22 415 434 AAGATGAGGCATAGCAGCAG 551979 4-10-6 54 22 416 435 GAAGATGAGGCATAGCAGCA 551916 2-10-8 44 24 416 435 GAAGATGAGGCATAGCAGCA 551948 3-10-7 59 24 416 435 GAAGATGAGGCATAGCAGCA 551980 4-10-6 49 24 417 436 AGAAGATGAGGCATAGCAGC 551917 2-10-8 48 26 417 436 AGAAGATGAGGCATAGCAGC 551949 3-10-7 60 26 417 436 AGAAGATGAGGCATAGCAGC 551981 4-10-6 57 26 418 437 AAGAAGATGAGGCATAGCAG 551918 2-10-8 53 28 418 437 AAGAAGATGAGGCATAGCAG 551950 3-10-7 57 28 418 437 AAGAAGATGAGGCATAGCAG 551982 4-10-6 57 28 687 706 CGAACCACTGAACAAATGGC 551919 2-10-8 65 39 687 706 CGAACCACTGAACAAATGGC 551951 3-10-7 57 39 687 706 CGAACCACTGAACAAATGGC 551983 4-10-6 53 39 1261 1280 TTCCGCAGTATGGATCGGCA 551920 2-10-8 57 719 1261 1280 TTCCGCAGTATGGATCGGCA 551952 3-10-7 67 719 1261 1280 TTCCGCAGTATGGATCGGCA 551984 4-10-6 62 719 1262 1281 GTTCCGCAGTATGGATCGGC 551921 2-10-8 60 212 1262 1281 GTTCCGCAGTATGGATCGGC 551953 3-10-7 57 212 1262 1281 GTTCCGCAGTATGGATCGGC 551985 4-10-6 58 212 1263 1282 AGTTCCGCAGTATGGATCGG 551922 2-10-8 63 720 1263 1282 AGTTCCGCAGTATGGATCGG 551954 3-10-7 61 720 1264 1283 GAGTTCCGCAGTATGGATCG 551923 2-10-8 50 721 1264 1283 GAGTTCCGCAGTATGGATCG 551955 3-10-7 44 721 1265 1284 GGAGTTCCGCAGTATGGATC 551924 2-10-8 52 1349 1265 1284 GGAGTTCCGCAGTATGGATC 551956 3-10-7 46 1349 1266 1285 AGGAGTTCCGCAGTATGGAT 551925 2-10-8 54 722 1266 1285 AGGAGTTCCGCAGTATGGAT 551957 3-10-7 51 722 1577 1596 GTGAAGCGAAGTGCACACGG 551926 2-10-8 70 224 1577 1596 GTGAAGCGAAGTGCACACGG 551958 3-10-7 72 224 1578 1597 GGTGAAGCGAAGTGCACACG 551927 2-10-8 60 801 1578 1597 GGTGAAGCGAAGTGCACACG 551959 3-10-7 61 801 1579 1598 AGGTGAAGCGAAGTGCACAC 551928 2-10-8 57 802 1579 1598 AGGTGAAGCGAAGTGCACAC 551960 3-10-7 58 802 1580 1599 GAGGTGAAGCGAAGTGCACA 551929 2-10-8 49 225 1580 1599 GAGGTGAAGCGAAGTGCACA 551961 3-10-7 26 225 1581 1600 AGAGGTGAAGCGAAGTGCAC 551930 2-10-8 54 804 1581 1600 AGAGGTGAAGCGAAGTGCAC 551962 3-10-7 57 804 1582 1601 CAGAGGTGAAGCGAAGTGCA 551931 2-10-8 46 805 1582 1601 CAGAGGTGAAGCGAAGTGCA 551963 3-10-7 56 805 1583 1602 GCAGAGGTGAAGCGAAGTGC 551932 2-10-8 57 226 1583 1602 GCAGAGGTGAAGCGAAGTGC 551964 3-10-7 53 226 1584 1603 TGCAGAGGTGAAGCGAAGTG 551933 2-10-8 65 806 1584 1603 TGCAGAGGTGAAGCGAAGTG 551965 3-10-7 54 806 1585 1604 GTGCAGAGGTGAAGCGAAGT 551934 2-10-8 58 807 1585 1604 GTGCAGAGGTGAAGCGAAGT 551966 3-10-7 69 807 1586 1605 CGTGCAGAGGTGAAGCGAAG 551935 2-10-8 63 227 1586 1605 CGTGCAGAGGTGAAGCGAAG 551967 3-10-7 53 227 1587 1606 ACGTGCAGAGGTGAAGCGAA 551936 2-10-8 67 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 551968 3-10-7 60 1350 1778 1797 AATTTATGCCTACAGCCTCC 551937 2-10-8 51 46 1778 1797 AATTTATGCCTACAGCCTCC 551969 3-10-7 42 46 1779 1798 CAATTTATGCCTACAGCCTC 551938 2-10-8 40 48 1779 1798 CAATTTATGCCTACAGCCTC 551970 3-10-7 38 48 1780 1799 CCAATTTATGCCTACAGCCT 551939 2-10-8 32 50 1780 1799 CCAATTTATGCCTACAGCCT 551971 3-10-7 46 50 1781 1800 ACCAATTTATGCCTACAGCC 551940 2-10-8 39 52 1781 1800 ACCAATTTATGCCTACAGCC 551972 3-10-7 51 52

TABLE 39 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 40 224 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 60 17 58 74 CTGGAGCCACCAGCAGG 552276 5-9-3 44 1288 59 75 ACTGGAGCCACCAGCAG 552277 5-9-3 39 1289 60 76 AACTGGAGCCACCAGCA 552278 5-9-3 37 1290 61 77 GAACTGGAGCCACCAGC 552279 5-9-3 50 1291 253 269 GAAGTCCACCACGAGTC 552280 5-9-3 2 9 254 270 AGAAGTCCACCACGAGT 552281 5-9-3 0 10 255 271 GAGAAGTCCACCACGAG 552282 5-9-3 13 11 256 272 AGAGAAGTCCACCACGA 552229 4-9-4 17 12 256 272 AGAGAAGTCCACCACGA 552283 5-9-3 27 12 411 427 GGCATAGCAGCAGGATG 552230 4-9-4 53 17 411 427 GGCATAGCAGCAGGATG 552284 5-9-3 0 17 412 428 AGGCATAGCAGCAGGAT 552231 4-9-4 31 18 412 428 AGGCATAGCAGCAGGAT 552285 5-9-3 56 18 413 429 GAGGCATAGCAGCAGGA 552232 4-9-4 35 19 413 429 GAGGCATAGCAGCAGGA 552286 5-9-3 43 19 414 430 TGAGGCATAGCAGCAGG 552233 4-9-4 40 21 414 430 TGAGGCATAGCAGCAGG 552287 5-9-3 44 21 415 431 ATGAGGCATAGCAGCAG 552234 4-9-4 0 23 415 431 ATGAGGCATAGCAGCAG 552288 5-9-3 44 23 416 432 GATGAGGCATAGCAGCA 552235 4-9-4 13 25 416 432 GATGAGGCATAGCAGCA 552289 5-9-3 21 25 417 433 AGATGAGGCATAGCAGC 552236 4-9-4 40 27 417 433 AGATGAGGCATAGCAGC 552290 5-9-3 34 27 418 434 AAGATGAGGCATAGCAG 552237 4-9-4 37 29 418 434 AAGATGAGGCATAGCAG 552291 5-9-3 34 29 670 686 ACTAGTAAACTGAGCCA 552239 4-9-4 58 1292 670 686 ACTAGTAAACTGAGCCA 552293 5-9-3 61 1292 671 687 CACTAGTAAACTGAGCC 552240 4-9-4 54 1293 671 687 CACTAGTAAACTGAGCC 552294 5-9-3 62 1293 672 688 GCACTAGTAAACTGAGC 552241 4-9-4 47 1294 672 688 GCACTAGTAAACTGAGC 552295 5-9-3 63 1294 687 703 ACCACTGAACAAATGGC 552242 4-9-4 61 40 687 703 ACCACTGAACAAATGGC 552296 5-9-3 61 40 688 704 AACCACTGAACAAATGG 552243 4-9-4 55 41 688 704 AACCACTGAACAAATGG 552297 5-9-3 52 41 689 705 GAACCACTGAACAAATG 552244 4-9-4 45 42 689 705 GAACCACTGAACAAATG 552298 5-9-3 27 42 690 706 CGAACCACTGAACAAAT 552245 4-9-4 41 43 690 706 CGAACCACTGAACAAAT 552299 5-9-3 32 43 1261 1277 CGCAGTATGGATCGGCA 552246 4-9-4 67 1295 1261 1277 CGCAGTATGGATCGGCA 552300 5-9-3 57 1295 1262 1278 CCGCAGTATGGATCGGC 552247 4-9-4 74 1296 1262 1278 CCGCAGTATGGATCGGC 552301 5-9-3 76 1296 1263 1279 TCCGCAGTATGGATCGG 552248 4-9-4 65 1297 1263 1279 TCCGCAGTATGGATCGG 552302 5-9-3 68 1297 1264 1280 TTCCGCAGTATGGATCG 552249 4-9-4 38 1298 1264 1280 TTCCGCAGTATGGATCG 552303 5-9-3 59 1298 1265 1281 GTTCCGCAGTATGGATC 552250 4-9-4 43 1299 1265 1281 GTTCCGCAGTATGGATC 552304 5-9-3 30 1299 1266 1282 AGTTCCGCAGTATGGAT 552251 4-9-4 52 1300 1266 1282 AGTTCCGCAGTATGGAT 552305 5-9-3 49 1300 1267 1283 GAGTTCCGCAGTATGGA 552252 4-9-4 51 1301 1267 1283 GAGTTCCGCAGTATGGA 552306 5-9-3 56 1301 1268 1284 GGAGTTCCGCAGTATGG 552253 4-9-4 47 1302 1268 1284 GGAGTTCCGCAGTATGG 552307 5-9-3 49 1302 1269 1285 AGGAGTTCCGCAGTATG 552254 4-9-4 50 1303 1577 1593 AAGCGAAGTGCACACGG 552255 4-9-4 64 1304 1578 1594 GAAGCGAAGTGCACACG 552256 4-9-4 57 1305 1579 1595 TGAAGCGAAGTGCACAC 552257 4-9-4 51 1306 1580 1596 GTGAAGCGAAGTGCACA 552258 4-9-4 62 1307 1581 1597 GGTGAAGCGAAGTGCAC 552259 4-9-4 59 1308 1582 1598 AGGTGAAGCGAAGTGCA 552260 4-9-4 56 1309 1583 1599 GAGGTGAAGCGAAGTGC 552261 4-9-4 54 1310 1584 1600 AGAGGTGAAGCGAAGTG 552262 4-9-4 47 1311 1585 1601 CAGAGGTGAAGCGAAGT 552263 4-9-4 45 1312 1586 1602 GCAGAGGTGAAGCGAAG 552264 4-9-4 52 1313 1587 1603 TGCAGAGGTGAAGCGAA 552265 4-9-4 58 1314 1588 1604 GTGCAGAGGTGAAGCGA 552266 4-9-4 54 1315 1589 1605 CGTGCAGAGGTGAAGCG 552267 4-9-4 43 1316 1590 1606 ACGTGCAGAGGTGAAGC 552268 4-9-4 57 1317 1778 1794 TTATGCCTACAGCCTCC 552269 4-9-4 34 47 1779 1795 TTTATGCCTACAGCCTC 552270 4-9-4 37 49 1780 1796 ATTTATGCCTACAGCCT 552271 4-9-4 42 51 1781 1797 AATTTATGCCTACAGCC 552272 4-9-4 36 53 1782 1798 CAATTTATGCCTACAGC 552273 4-9-4 25 54 1783 1799 CCAATTTATGCCTACAG 552274 4-9-4 11 55 1784 1800 ACCAATTTATGCCTACA 552275 4-9-4 38 56

TABLE 40 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 38 1354 414 429 GAGGCATAGCAGCAGG 509959 3-10-3 49 145 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 55 17 58 73 TGGAGCCACCAGCAGG 552384 2-9-5 41 1318 58 73 TGGAGCCACCAGCAGG 552440 3-9-4 57 1318 59 74 CTGGAGCCACCAGCAG 552385 2-9-5 53 1319 59 74 CTGGAGCCACCAGCAG 552441 3-9-4 38 1319 60 75 ACTGGAGCCACCAGCA 552386 2-9-5 42 1320 60 75 ACTGGAGCCACCAGCA 552442 3-9-4 72 1320 61 76 AACTGGAGCCACCAGC 552387 2-9-5 43 1321 61 76 AACTGGAGCCACCAGC 552443 3-9-4 56 1321 62 77 GAACTGGAGCCACCAG 552388 2-9-5 18 86 62 77 GAACTGGAGCCACCAG 552444 3-9-4 39 86 411 426 GCATAGCAGCAGGATG 552389 2-9-5 24 137 411 426 GCATAGCAGCAGGATG 552445 3-9-4 53 137 412 427 GGCATAGCAGCAGGAT 552390 2-9-5 40 140 412 427 GGCATAGCAGCAGGAT 552446 3-9-4 57 140 413 428 AGGCATAGCAGCAGGA 552391 2-9-5 51 143 413 428 AGGCATAGCAGCAGGA 552447 3-9-4 53 143 414 429 GAGGCATAGCAGCAGG 552392 2-9-5 0 145 414 429 GAGGCATAGCAGCAGG 552448 3-9-4 57 145 415 430 TGAGGCATAGCAGCAG 552393 2-9-5 52 147 415 430 TGAGGCATAGCAGCAG 552449 3-9-4 49 147 416 431 ATGAGGCATAGCAGCA 552394 2-9-5 32 149 416 431 ATGAGGCATAGCAGCA 552450 3-9-4 44 149 417 432 GATGAGGCATAGCAGC 552395 2-9-5 33 151 417 432 GATGAGGCATAGCAGC 552451 3-9-4 38 151 418 433 AGATGAGGCATAGCAG 552396 2-9-5 46 153 418 433 AGATGAGGCATAGCAG 552452 3-9-4 30 153 457 473 ACGGGCAACATACCTTG 552130 2-9-6 46 33 457 473 ACGGGCAACATACCTTG 552184 3-9-5 34 33 457 473 ACGGGCAACATACCTTG 552238 4-9-4 41 33 457 473 ACGGGCAACATACCTTG 552292 5-9-3 45 33 457 473 ACGGGCAACATACCTTG 552346 6-9-2 0 33 457 472 CGGGCAACATACCTTG 552397 2-9-5 37 167 457 472 CGGGCAACATACCTTG 552453 3-9-4 45 167 458 473 ACGGGCAACATACCTT 552398 2-9-5 42 168 458 473 ACGGGCAACATACCTT 552454 3-9-4 39 168 670 685 CTAGTAAACTGAGCCA 552399 2-9-5 34 181 671 686 ACTAGTAAACTGAGCC 552400 2-9-5 47 1322 672 687 CACTAGTAAACTGAGC 552401 2-9-5 53 1323 673 688 GCACTAGTAAACTGAG 552402 2-9-5 47 1324 687 702 CCACTGAACAAATGGC 552403 2-9-5 70 188 688 703 ACCACTGAACAAATGG 552404 2-9-5 44 190 689 704 AACCACTGAACAAATG 552405 2-9-5 0 191 690 705 GAACCACTGAACAAAT 552406 2-9-5 25 192 691 706 CGAACCACTGAACAAA 552407 2-9-5 23 194 1261 1276 GCAGTATGGATCGGCA 552408 2-9-5 73 211 1262 1277 CGCAGTATGGATCGGC 552409 2-9-5 71 1325 1263 1278 CCGCAGTATGGATCGG 552410 2-9-5 52 1326 1264 1279 TCCGCAGTATGGATCG 552411 2-9-5 62 1327 1265 1280 TTCCGCAGTATGGATC 552412 2-9-5 50 1328 1266 1281 GTTCCGCAGTATGGAT 552413 2-9-5 55 1329 1267 1282 AGTTCCGCAGTATGGA 552414 2-9-5 64 1330 1268 1283 GAGTTCCGCAGTATGG 552415 2-9-5 45 1331 1269 1284 GGAGTTCCGCAGTATG 552416 2-9-5 45 1332 1270 1285 AGGAGTTCCGCAGTAT 552417 2-9-5 37 1333 1577 1592 AGCGAAGTGCACACGG 552418 2-9-5 73 1334 1578 1593 AAGCGAAGTGCACACG 552419 2-9-5 68 1335 1579 1594 GAAGCGAAGTGCACAC 552420 2-9-5 64 1336 1580 1595 TGAAGCGAAGTGCACA 552421 2-9-5 54 1337 1581 1596 GTGAAGCGAAGTGCAC 552422 2-9-5 60 1338 1582 1597 GGTGAAGCGAAGTGCA 552423 2-9-5 62 1339 1583 1598 AGGTGAAGCGAAGTGC 552424 2-9-5 60 1340 1584 1599 GAGGTGAAGCGAAGTG 552425 2-9-5 46 1341 1585 1600 AGAGGTGAAGCGAAGT 552426 2-9-5 48 1342 1586 1601 CAGAGGTGAAGCGAAG 552427 2-9-5 36 1343 1587 1602 GCAGAGGTGAAGCGAA 552428 2-9-5 57 1344 1588 1603 TGCAGAGGTGAAGCGA 552429 2-9-5 36 1345 1589 1604 GTGCAGAGGTGAAGCG 552430 2-9-5 42 1346 1590 1605 CGTGCAGAGGTGAAGC 552431 2-9-5 60 1347 1591 1606 ACGTGCAGAGGTGAAG 552432 2-9-5 44 1348 1778 1793 TATGCCTACAGCCTCC 552433 2-9-5 55 230 1779 1794 TTATGCCTACAGCCTC 552434 2-9-5 46 231 1780 1795 TTTATGCCTACAGCCT 552435 2-9-5 47 232 1781 1796 ATTTATGCCTACAGCC 552436 2-9-5 25 233 1782 1797 AATTTATGCCTACAGC 552437 2-9-5 19 234 1783 1798 CAATTTATGCCTACAG 552438 2-9-5 25 235 1784 1799 CCAATTTATGCCTACA 552439 2-9-5 22 236

TABLE 41 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 414 429 GAGGCATAGCAGCAGG 509959 3-10-3 49 145 58 73 TGGAGCCACCAGCAGG 552496 4-9-3 35 1318 59 74 CTGGAGCCACCAGCAG 552497 4-9-3 60 1319 60 75 ACTGGAGCCACCAGCA 552498 4-9-3 20 1320 61 76 AACTGGAGCCACCAGC 552499 4-9-3 45 1321 62 77 GAACTGGAGCCACCAG 552500 4-9-3 53 86 411 426 GCATAGCAGCAGGATG 552501 4-9-3 56 137 412 427 GGCATAGCAGCAGGAT 552502 4-9-3 50 140 413 428 AGGCATAGCAGCAGGA 552503 4-9-3 36 143 414 429 GAGGCATAGCAGCAGG 552504 4-9-3 50 145 415 430 TGAGGCATAGCAGCAG 552505 4-9-3 53 147 416 431 ATGAGGCATAGCAGCA 552506 4-9-3 49 149 417 432 GATGAGGCATAGCAGC 552507 4-9-3 35 151 418 433 AGATGAGGCATAGCAG 552508 4-9-3 62 153 457 472 CGGGCAACATACCTTG 552509 4-9-3 65 167 458 473 ACGGGCAACATACCTT 552510 4-9-3 54 168 670 685 CTAGTAAACTGAGCCA 552455 3-9-4 60 181 670 685 CTAGTAAACTGAGCCA 552511 4-9-3 65 181 671 686 ACTAGTAAACTGAGCC 552456 3-9-4 69 1322 671 686 ACTAGTAAACTGAGCC 552512 4-9-3 63 1322 672 687 CACTAGTAAACTGAGC 552457 3-9-4 4 1323 672 687 CACTAGTAAACTGAGC 552513 4-9-3 50 1323 673 688 GCACTAGTAAACTGAG 552458 3-9-4 59 1324 673 688 GCACTAGTAAACTGAG 552514 4-9-3 53 1324 687 702 CCACTGAACAAATGGC 552459 3-9-4 69 188 687 702 CCACTGAACAAATGGC 552515 4-9-3 68 188 688 703 ACCACTGAACAAATGG 552460 3-9-4 3 190 688 703 ACCACTGAACAAATGG 552516 4-9-3 65 190 689 704 AACCACTGAACAAATG 552461 3-9-4 37 191 689 704 AACCACTGAACAAATG 552517 4-9-3 54 191 690 705 GAACCACTGAACAAAT 552462 3-9-4 42 192 690 705 GAACCACTGAACAAAT 552518 4-9-3 23 192 691 706 CGAACCACTGAACAAA 552463 3-9-4 28 194 691 706 CGAACCACTGAACAAA 552519 4-9-3 32 194 1261 1276 GCAGTATGGATCGGCA 552464 3-9-4 72 211 1261 1276 GCAGTATGGATCGGCA 552520 4-9-3 61 211 1262 1277 CGCAGTATGGATCGGC 552465 3-9-4 68 1325 1262 1277 CGCAGTATGGATCGGC 552521 4-9-3 68 1325 1263 1278 CCGCAGTATGGATCGG 552466 3-9-4 76 1326 1263 1278 CCGCAGTATGGATCGG 552522 4-9-3 71 1326 1264 1279 TCCGCAGTATGGATCG 552467 3-9-4 72 1327 1264 1279 TCCGCAGTATGGATCG 552523 4-9-3 73 1327 1265 1280 TTCCGCAGTATGGATC 552468 3-9-4 50 1328 1265 1280 TTCCGCAGTATGGATC 552524 4-9-3 49 1328 1266 1281 GTTCCGCAGTATGGAT 552469 3-9-4 65 1329 1266 1281 GTTCCGCAGTATGGAT 552525 4-9-3 45 1329 1267 1282 AGTTCCGCAGTATGGA 552470 3-9-4 58 1330 1267 1282 AGTTCCGCAGTATGGA 552526 4-9-3 39 1330 1268 1283 GAGTTCCGCAGTATGG 552471 3-9-4 30 1331 1268 1283 GAGTTCCGCAGTATGG 552527 4-9-3 39 1331 1269 1284 GGAGTTCCGCAGTATG 552472 3-9-4 43 1332 1269 1284 GGAGTTCCGCAGTATG 552528 4-9-3 43 1332 1270 1285 AGGAGTTCCGCAGTAT 552473 3-9-4 25 1333 1270 1285 AGGAGTTCCGCAGTAT 552529 4-9-3 50 1333 1577 1592 AGCGAAGTGCACACGG 552474 3-9-4 70 1334 1577 1592 AGCGAAGTGCACACGG 552530 4-9-3 73 1334 1578 1593 AAGCGAAGTGCACACG 552475 3-9-4 64 1335 1578 1593 AAGCGAAGTGCACACG 552531 4-9-3 62 1335 1579 1594 GAAGCGAAGTGCACAC 552476 3-9-4 50 1336 1580 1595 TGAAGCGAAGTGCACA 552477 3-9-4 66 1337 1581 1596 GTGAAGCGAAGTGCAC 552478 3-9-4 68 1338 1582 1597 GGTGAAGCGAAGTGCA 552479 3-9-4 60 1339 1583 1598 AGGTGAAGCGAAGTGC 552480 3-9-4 58 1340 1584 1599 GAGGTGAAGCGAAGTG 552481 3-9-4 54 1341 1585 1600 AGAGGTGAAGCGAAGT 552482 3-9-4 44 1342 1586 1601 CAGAGGTGAAGCGAAG 552483 3-9-4 17 1343 1587 1602 GCAGAGGTGAAGCGAA 552484 3-9-4 64 1344 1588 1603 TGCAGAGGTGAAGCGA 552485 3-9-4 56 1345 1589 1604 GTGCAGAGGTGAAGCG 552486 3-9-4 26 1346 1590 1605 CGTGCAGAGGTGAAGC 552487 3-9-4 42 1347 1591 1606 ACGTGCAGAGGTGAAG 552488 3-9-4 35 1348 1778 1793 TATGCCTACAGCCTCC 552489 3-9-4 46 230 1779 1794 TTATGCCTACAGCCTC 552490 3-9-4 41 231 1780 1795 TTTATGCCTACAGCCT 552491 3-9-4 38 232 1781 1796 ATTTATGCCTACAGCC 552492 3-9-4 47 233 1782 1797 AATTTATGCCTACAGC 552493 3-9-4 49 234 1783 1798 CAATTTATGCCTACAG 552494 3-9-4 22 235 1784 1799 CCAATTTATGCCTACA 552495 3-9-4 0 236

TABLE 42 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 56 224 55 414 429 GAGGCATAGCAGCAGG 509959 3-10-3 54 145 58 73 TGGAGCCACCAGCAGG 552552 5-9-2 32 1355 59 74 CTGGAGCCACCAGCAG 552553 5-9-2 53 1319 60 75 ACTGGAGCCACCAGCA 552554 5-9-2 48 1320 61 76 AACTGGAGCCACCAGC 552555 5-9-2 39 1321 62 77 GAACTGGAGCCACCAG 552556 5-9-2 39 86 411 426 GCATAGCAGCAGGATG 552557 5-9-2 54 137 412 427 GGCATAGCAGCAGGAT 552558 5-9-2 41 140 413 428 AGGCATAGCAGCAGGA 552559 5-9-2 56 143 414 429 GAGGCATAGCAGCAGG 552560 5-9-2 39 145 415 430 TGAGGCATAGCAGCAG 552561 5-9-2 51 147 416 431 ATGAGGCATAGCAGCA 552562 5-9-2 56 149 417 432 GATGAGGCATAGCAGC 552563 5-9-2 31 151 418 433 AGATGAGGCATAGCAG 552564 5-9-2 31 153 457 472 CGGGCAACATACCTTG 552565 5-9-2 53 167 458 473 ACGGGCAACATACCTT 552566 5-9-2 46 168 670 685 CTAGTAAACTGAGCCA 552567 5-9-2 63 181 671 686 ACTAGTAAACTGAGCC 552568 5-9-2 66 1322 672 687 CACTAGTAAACTGAGC 552569 5-9-2 60 1323 673 688 GCACTAGTAAACTGAG 552570 5-9-2 60 1324 687 702 CCACTGAACAAATGGC 552571 5-9-2 44 188 688 703 ACCACTGAACAAATGG 552572 5-9-2 52 190 689 704 AACCACTGAACAAATG 552573 5-9-2 20 191 690 705 GAACCACTGAACAAAT 552574 5-9-2 36 192 691 706 CGAACCACTGAACAAA 552575 5-9-2 19 194 1261 1276 GCAGTATGGATCGGCA 552576 5-9-2 61 211 1262 1277 CGCAGTATGGATCGGC 552577 5-9-2 57 1325 1263 1278 CCGCAGTATGGATCGG 552578 5-9-2 71 1326 1264 1279 TCCGCAGTATGGATCG 552579 5-9-2 59 1327 1265 1280 TTCCGCAGTATGGATC 552580 5-9-2 58 1328 1266 1281 GTTCCGCAGTATGGAT 552581 5-9-2 51 1329 1267 1282 AGTTCCGCAGTATGGA 552582 5-9-2 40 1330 1268 1283 GAGTTCCGCAGTATGG 552583 5-9-2 35 1331 1269 1284 GGAGTTCCGCAGTATG 552584 5-9-2 50 1332 1270 1285 AGGAGTTCCGCAGTAT 552585 5-9-2 48 1333 1577 1592 AGCGAAGTGCACACGG 552586 5-9-2 74 1334 1578 1593 AAGCGAAGTGCACACG 552587 5-9-2 68 1335 1579 1594 GAAGCGAAGTGCACAC 552532 4-9-3 59 1336 1579 1594 GAAGCGAAGTGCACAC 552588 5-9-2 67 1336 1580 1595 TGAAGCGAAGTGCACA 552533 4-9-3 52 1337 1580 1595 TGAAGCGAAGTGCACA 552589 5-9-2 47 1337 1581 1596 GTGAAGCGAAGTGCAC 552534 4-9-3 71 1338 1581 1596 GTGAAGCGAAGTGCAC 552590 5-9-2 58 1338 1582 1597 GGTGAAGCGAAGTGCA 552535 4-9-3 59 1339 1582 1597 GGTGAAGCGAAGTGCA 552591 5-9-2 46 1339 1583 1598 AGGTGAAGCGAAGTGC 552536 4-9-3 19 1340 1583 1598 AGGTGAAGCGAAGTGC 552592 5-9-2 44 1340 1584 1599 GAGGTGAAGCGAAGTG 552537 4-9-3 26 1341 1584 1599 GAGGTGAAGCGAAGTG 552593 5-9-2 39 1341 1585 1600 AGAGGTGAAGCGAAGT 552538 4-9-3 54 1342 1585 1600 AGAGGTGAAGCGAAGT 552594 5-9-2 52 1342 1586 1601 CAGAGGTGAAGCGAAG 552539 4-9-3 50 1343 1586 1601 CAGAGGTGAAGCGAAG 552595 5-9-2 57 1343 1587 1602 GCAGAGGTGAAGCGAA 552540 4-9-3 60 1344 1587 1602 GCAGAGGTGAAGCGAA 552596 5-9-2 58 1344 1588 1603 TGCAGAGGTGAAGCGA 552541 4-9-3 68 1345 1588 1603 TGCAGAGGTGAAGCGA 552597 5-9-2 52 1345 1589 1604 GTGCAGAGGTGAAGCG 552542 4-9-3 63 1346 1589 1604 GTGCAGAGGTGAAGCG 552598 5-9-2 51 1346 1590 1605 CGTGCAGAGGTGAAGC 552543 4-9-3 44 1347 1590 1605 CGTGCAGAGGTGAAGC 552600 5-9-2 51 1347 1591 1606 ACGTGCAGAGGTGAAG 552544 4-9-3 45 1348 1591 1606 ACGTGCAGAGGTGAAG 552602 5-9-2 13 1348 1778 1793 TATGCCTACAGCCTCC 552545 4-9-3 42 230 1778 1793 TATGCCTACAGCCTCC 552604 5-9-2 42 230 1779 1794 TTATGCCTACAGCCTC 552546 4-9-3 46 231 1779 1794 TTATGCCTACAGCCTC 552606 5-9-2 42 231 1780 1795 TTTATGCCTACAGCCT 552547 4-9-3 38 232 1780 1795 TTTATGCCTACAGCCT 552608 5-9-2 37 232 1781 1796 ATTTATGCCTACAGCC 552548 4-9-3 49 233 1781 1796 ATTTATGCCTACAGCC 552610 5-9-2 41 233 1782 1797 AATTTATGCCTACAGC 552549 4-9-3 34 234 1782 1797 AATTTATGCCTACAGC 552612 5-9-2 23 234 1783 1798 CAATTTATGCCTACAG 552550 4-9-3 13 235 1783 1798 CAATTTATGCCTACAG 552614 5-9-2 11 235 1784 1799 CCAATTTATGCCTACA 552551 4-9-3 8 236 1784 1799 CCAATTTATGCCTACA 552616 5-9-2 6 236

TABLE 43 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 47 224 1780 1799 CCAATTTATGCCTACAGCCT 509934 5-10-5 67 50 58 77 GAACTGGAGCCACCAGCAGG 552007 6-10-4 53 83 58 77 GAACTGGAGCCACCAGCAGG 552039 7-10-3 74 83 253 272 AGAGAAGTCCACCACGAGTC 552008 6-10-4 47 103 253 272 AGAGAAGTCCACCACGAGTC 552040 7-10-3 57 103 411 430 TGAGGCATAGCAGCAGGATG 552009 6-10-4 70 136 411 430 TGAGGCATAGCAGCAGGATG 552041 7-10-3 65 136 412 431 ATGAGGCATAGCAGCAGGAT 552010 6-10-4 51 139 412 431 ATGAGGCATAGCAGCAGGAT 552042 7-10-3 59 139 413 432 GATGAGGCATAGCAGCAGGA 552011 6-10-4 47 142 413 432 GATGAGGCATAGCAGCAGGA 552043 7-10-3 36 142 414 433 AGATGAGGCATAGCAGCAGG 552012 6-10-4 62 20 414 433 AGATGAGGCATAGCAGCAGG 552044 7-10-3 82 20 415 434 AAGATGAGGCATAGCAGCAG 552013 6-10-4 72 22 415 434 AAGATGAGGCATAGCAGCAG 552045 7-10-3 62 22 416 435 GAAGATGAGGCATAGCAGCA 552014 6-10-4 73 24 416 435 GAAGATGAGGCATAGCAGCA 552046 7-10-3 74 24 417 436 AGAAGATGAGGCATAGCAGC 552015 6-10-4 66 26 417 436 AGAAGATGAGGCATAGCAGC 552047 7-10-3 60 26 418 437 AAGAAGATGAGGCATAGCAG 552016 6-10-4 67 28 418 437 AAGAAGATGAGGCATAGCAG 552048 7-10-3 60 28 687 706 CGAACCACTGAACAAATGGC 552017 6-10-4 72 39 687 706 CGAACCACTGAACAAATGGC 552049 7-10-3 68 39 1261 1280 TTCCGCAGTATGGATCGGCA 552018 6-10-4 89 719 1261 1280 TTCCGCAGTATGGATCGGCA 552050 7-10-3 86 719 1262 1281 GTTCCGCAGTATGGATCGGC 552019 6-10-4 87 212 1262 1281 GTTCCGCAGTATGGATCGGC 552051 7-10-3 86 212 1263 1282 AGTTCCGCAGTATGGATCGG 551986 4-10-6 64 720 1263 1282 AGTTCCGCAGTATGGATCGG 552020 6-10-4 86 720 1263 1282 AGTTCCGCAGTATGGATCGG 552052 7-10-3 87 720 1264 1283 GAGTTCCGCAGTATGGATCG 551987 4-10-6 76 721 1264 1283 GAGTTCCGCAGTATGGATCG 552021 6-10-4 84 721 1264 1283 GAGTTCCGCAGTATGGATCG 552053 7-10-3 75 721 1265 1284 GGAGTTCCGCAGTATGGATC 551988 4-10-6 5 1349 1265 1284 GGAGTTCCGCAGTATGGATC 552005 5-10-5 72 1349 1265 1284 GGAGTTCCGCAGTATGGATC 552022 6-10-4 80 1349 1265 1284 GGAGTTCCGCAGTATGGATC 552054 7-10-3 83 1349 1266 1285 AGGAGTTCCGCAGTATGGAT 551989 4-10-6 64 722 1266 1285 AGGAGTTCCGCAGTATGGAT 552023 6-10-4 78 722 1266 1285 AGGAGTTCCGCAGTATGGAT 552055 7-10-3 57 722 1577 1596 GTGAAGCGAAGTGCACACGG 551990 4-10-6 83 224 1577 1596 GTGAAGCGAAGTGCACACGG 552024 6-10-4 89 224 1577 1596 GTGAAGCGAAGTGCACACGG 552056 7-10-3 82 224 1578 1597 GGTGAAGCGAAGTGCACACG 551991 4-10-6 0 801 1578 1597 GGTGAAGCGAAGTGCACACG 552025 6-10-4 89 801 1578 1597 GGTGAAGCGAAGTGCACACG 552057 7-10-3 89 801 1579 1598 AGGTGAAGCGAAGTGCACAC 551992 4-10-6 67 802 1579 1598 AGGTGAAGCGAAGTGCACAC 552026 6-10-4 84 802 1579 1598 AGGTGAAGCGAAGTGCACAC 552058 7-10-3 82 802 1580 1599 GAGGTGAAGCGAAGTGCACA 551993 4-10-6 78 225 1580 1599 GAGGTGAAGCGAAGTGCACA 552027 6-10-4 85 225 1580 1599 GAGGTGAAGCGAAGTGCACA 552059 7-10-3 85 225 1581 1600 AGAGGTGAAGCGAAGTGCAC 551994 4-10-6 82 804 1581 1600 AGAGGTGAAGCGAAGTGCAC 552028 6-10-4 82 804 1581 1600 AGAGGTGAAGCGAAGTGCAC 552060 7-10-3 74 804 1582 1601 CAGAGGTGAAGCGAAGTGCA 551995 4-10-6 81 805 1582 1601 CAGAGGTGAAGCGAAGTGCA 552029 6-10-4 81 805 1582 1601 CAGAGGTGAAGCGAAGTGCA 552061 7-10-3 81 805 1583 1602 GCAGAGGTGAAGCGAAGTGC 551996 4-10-6 79 226 1583 1602 GCAGAGGTGAAGCGAAGTGC 552030 6-10-4 86 226 1583 1602 GCAGAGGTGAAGCGAAGTGC 552062 7-10-3 85 226 1584 1603 TGCAGAGGTGAAGCGAAGTG 551997 4-10-6 80 806 1584 1603 TGCAGAGGTGAAGCGAAGTG 552031 6-10-4 86 806 1585 1604 GTGCAGAGGTGAAGCGAAGT 551998 4-10-6 74 807 1585 1604 GTGCAGAGGTGAAGCGAAGT 552032 6-10-4 78 807 1586 1605 CGTGCAGAGGTGAAGCGAAG 551999 4-10-6 79 227 1586 1605 CGTGCAGAGGTGAAGCGAAG 552033 6-10-4 80 227 1587 1606 ACGTGCAGAGGTGAAGCGAA 552000 4-10-6 84 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 552006 5-10-5 86 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 552034 6-10-4 81 1350 1778 1797 AATTTATGCCTACAGCCTCC 552001 4-10-6 66 46 1778 1797 AATTTATGCCTACAGCCTCC 552035 6-10-4 55 46 1779 1798 CAATTTATGCCTACAGCCTC 552002 4-10-6 54 48 1779 1798 CAATTTATGCCTACAGCCTC 552036 6-10-4 58 48 1780 1799 CCAATTTATGCCTACAGCCT 552003 4-10-6 50 50 1780 1799 CCAATTTATGCCTACAGCCT 552037 6-10-4 43 50 1781 1800 ACCAATTTATGCCTACAGCC 552004 4-10-6 56 52 1781 1800 ACCAATTTATGCCTACAGCC 552038 6-10-4 66 52

TABLE 44 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 61 224 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 66 17 58 74 CTGGAGCCACCAGCAGG 552168 3-9-5 64 1288 58 74 CTGGAGCCACCAGCAGG 552222 4-9-4 76 1288 59 75 ACTGGAGCCACCAGCAG 552169 3-9-5 65 1289 59 75 ACTGGAGCCACCAGCAG 552223 4-9-4 41 1289 60 76 AACTGGAGCCACCAGCA 552170 3-9-5 58 1290 60 76 AACTGGAGCCACCAGCA 552224 4-9-4 58 1290 61 77 GAACTGGAGCCACCAGC 552171 3-9-5 51 1291 61 77 GAACTGGAGCCACCAGC 552225 4-9-4 49 1291 253 269 GAAGTCCACCACGAGTC 552172 3-9-5 23 9 253 269 GAAGTCCACCACGAGTC 552226 4-9-4 36 9 254 270 AGAAGTCCACCACGAGT 552173 3-9-5 44 10 254 270 AGAAGTCCACCACGAGT 552227 4-9-4 20 10 255 271 GAGAAGTCCACCACGAG 552174 3-9-5 28 11 255 271 GAGAAGTCCACCACGAG 552228 4-9-4 29 11 256 272 AGAGAAGTCCACCACGA 552175 3-9-5 56 12 411 427 GGCATAGCAGCAGGATG 552176 3-9-5 66 17 412 428 AGGCATAGCAGCAGGAT 552177 3-9-5 53 18 413 429 GAGGCATAGCAGCAGGA 552178 3-9-5 57 19 414 430 TGAGGCATAGCAGCAGG 552179 3-9-5 56 21 415 431 ATGAGGCATAGCAGCAG 552180 3-9-5 51 23 416 432 GATGAGGCATAGCAGCA 552181 3-9-5 51 25 417 433 AGATGAGGCATAGCAGC 552182 3-9-5 63 27 418 434 AAGATGAGGCATAGCAG 552183 3-9-5 60 29 670 686 ACTAGTAAACTGAGCCA 552185 3-9-5 67 1292 671 687 CACTAGTAAACTGAGCC 552186 3-9-5 37 1293 672 688 GCACTAGTAAACTGAGC 552187 3-9-5 68 1294 687 703 ACCACTGAACAAATGGC 552188 3-9-5 71 40 688 704 AACCACTGAACAAATGG 552189 3-9-5 51 41 689 705 GAACCACTGAACAAATG 552190 3-9-5 47 42 690 706 CGAACCACTGAACAAAT 552191 3-9-5 50 43 1261 1277 CGCAGTATGGATCGGCA 552192 3-9-5 80 1295 1262 1278 CCGCAGTATGGATCGGC 552193 3-9-5 73 1296 1263 1279 TCCGCAGTATGGATCGG 552194 3-9-5 58 1297 1264 1280 TTCCGCAGTATGGATCG 552195 3-9-5 60 1298 1265 1281 GTTCCGCAGTATGGATC 552196 3-9-5 54 1299 1266 1282 AGTTCCGCAGTATGGAT 552197 3-9-5 64 1300 1267 1283 GAGTTCCGCAGTATGGA 552198 3-9-5 62 1301 1268 1284 GGAGTTCCGCAGTATGG 552199 3-9-5 57 1302 1269 1285 AGGAGTTCCGCAGTATG 552200 3-9-5 52 1303 1577 1593 AAGCGAAGTGCACACGG 552201 3-9-5 73 1304 1578 1594 GAAGCGAAGTGCACACG 552202 3-9-5 60 1305 1579 1595 TGAAGCGAAGTGCACAC 552203 3-9-5 60 1306 1580 1596 GTGAAGCGAAGTGCACA 552204 3-9-5 63 1307 1581 1597 GGTGAAGCGAAGTGCAC 552151 2-9-6 71 1308 1581 1597 GGTGAAGCGAAGTGCAC 552205 3-9-5 64 1308 1582 1598 AGGTGAAGCGAAGTGCA 552152 2-9-6 69 1309 1582 1598 AGGTGAAGCGAAGTGCA 552206 3-9-5 71 1309 1583 1599 GAGGTGAAGCGAAGTGC 552153 2-9-6 63 1310 1583 1599 GAGGTGAAGCGAAGTGC 552207 3-9-5 71 1310 1584 1600 AGAGGTGAAGCGAAGTG 552154 2-9-6 56 1311 1584 1600 AGAGGTGAAGCGAAGTG 552208 3-9-5 52 1311 1585 1601 CAGAGGTGAAGCGAAGT 552155 2-9-6 61 1312 1585 1601 CAGAGGTGAAGCGAAGT 552209 3-9-5 50 1312 1586 1602 GCAGAGGTGAAGCGAAG 552156 2-9-6 40 1313 1586 1602 GCAGAGGTGAAGCGAAG 552210 3-9-5 66 1313 1587 1603 TGCAGAGGTGAAGCGAA 552157 2-9-6 45 1314 1587 1603 TGCAGAGGTGAAGCGAA 552211 3-9-5 63 1314 1588 1604 GTGCAGAGGTGAAGCGA 552158 2-9-6 66 1315 1588 1604 GTGCAGAGGTGAAGCGA 552212 3-9-5 62 1315 1589 1605 CGTGCAGAGGTGAAGCG 552159 2-9-6 68 1316 1589 1605 CGTGCAGAGGTGAAGCG 552213 3-9-5 64 1316 1590 1606 ACGTGCAGAGGTGAAGC 552160 2-9-6 78 1317 1590 1606 ACGTGCAGAGGTGAAGC 552214 3-9-5 72 1317 1778 1794 TTATGCCTACAGCCTCC 552161 2-9-6 57 47 1778 1794 TTATGCCTACAGCCTCC 552215 3-9-5 54 47 1779 1795 TTTATGCCTACAGCCTC 552162 2-9-6 54 49 1779 1795 TTTATGCCTACAGCCTC 552216 3-9-5 49 49 1780 1796 ATTTATGCCTACAGCCT 552163 2-9-6 65 51 1780 1796 ATTTATGCCTACAGCCT 552217 3-9-5 50 51 1781 1797 AATTTATGCCTACAGCC 552164 2-9-6 48 53 1781 1797 AATTTATGCCTACAGCC 552218 3-9-5 39 53 1782 1798 CAATTTATGCCTACAGC 552165 2-9-6 46 54 1782 1798 CAATTTATGCCTACAGC 552219 3-9-5 41 54 1783 1799 CCAATTTATGCCTACAG 552166 2-9-6 42 55 1783 1799 CCAATTTATGCCTACAG 552220 3-9-5 32 55 1784 1800 ACCAATTTATGCCTACA 552167 2-9-6 47 56 1784 1800 ACCAATTTATGCCTACA 552221 3-9-5 33 56

TABLE 45 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 87 224 56 1780 1799 CCAATTTATGCCTACAGCCT 509934 5-10-5 56 50 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 69 17 58 77 GAACTGGAGCCACCAGCAGG 552071 8-10-2 73 83 58 74 CTGGAGCCACCAGCAGG 552114 2-9-6 64 1288 59 75 ACTGGAGCCACCAGCAG 552115 2-9-6 61 1289 60 76 AACTGGAGCCACCAGCA 552116 2-9-6 53 1290 61 77 GAACTGGAGCCACCAGC 552117 2-9-6 69 1291 253 272 AGAGAAGTCCACCACGAGTC 552072 8-10-2 39 103 253 269 GAAGTCCACCACGAGTC 552118 2-9-6 49 9 254 270 AGAAGTCCACCACGAGT 552119 2-9-6 49 10 255 271 GAGAAGTCCACCACGAG 552120 2-9-6 21 11 256 272 AGAGAAGTCCACCACGA 552121 2-9-6 27 12 411 430 TGAGGCATAGCAGCAGGATG 552073 8-10-2 73 136 411 427 GGCATAGCAGCAGGATG 552122 2-9-6 48 17 412 431 ATGAGGCATAGCAGCAGGAT 552074 8-10-2 69 139 412 428 AGGCATAGCAGCAGGAT 552123 2-9-6 68 18 413 432 GATGAGGCATAGCAGCAGGA 552075 8-10-2 78 142 413 429 GAGGCATAGCAGCAGGA 552124 2-9-6 47 19 414 433 AGATGAGGCATAGCAGCAGG 552076 8-10-2 63 20 414 430 TGAGGCATAGCAGCAGG 552125 2-9-6 72 21 415 434 AAGATGAGGCATAGCAGCAG 552077 8-10-2 62 22 415 431 ATGAGGCATAGCAGCAG 552126 2-9-6 64 23 416 435 GAAGATGAGGCATAGCAGCA 552078 8-10-2 59 24 416 432 GATGAGGCATAGCAGCA 552127 2-9-6 65 25 417 436 AGAAGATGAGGCATAGCAGC 552079 8-10-2 80 26 417 433 AGATGAGGCATAGCAGC 552128 2-9-6 78 27 418 437 AAGAAGATGAGGCATAGCAG 552080 8-10-2 74 28 418 434 AAGATGAGGCATAGCAG 552129 2-9-6 68 29 457 473 ACGGGCAACATACCTTG 552130 2-9-6 46 33 670 686 ACTAGTAAACTGAGCCA 552131 2-9-6 61 1292 671 687 CACTAGTAAACTGAGCC 552132 2-9-6 66 1293 672 688 GCACTAGTAAACTGAGC 552133 2-9-6 78 1294 687 706 CGAACCACTGAACAAATGGC 552081 8-10-2 69 39 687 703 ACCACTGAACAAATGGC 552134 2-9-6 68 40 688 704 AACCACTGAACAAATGG 552135 2-9-6 59 41 689 705 GAACCACTGAACAAATG 552136 2-9-6 39 42 690 706 CGAACCACTGAACAAAT 552137 2-9-6 36 43 1261 1280 TTCCGCAGTATGGATCGGCA 552082 8-10-2 86 719 1261 1277 CGCAGTATGGATCGGCA 552138 2-9-6 80 1295 1262 1281 GTTCCGCAGTATGGATCGGC 552083 8-10-2 85 212 1262 1278 CCGCAGTATGGATCGGC 552139 2-9-6 80 1296 1263 1282 AGTTCCGCAGTATGGATCGG 552084 8-10-2 86 720 1263 1279 TCCGCAGTATGGATCGG 552140 2-9-6 70 1297 1264 1283 GAGTTCCGCAGTATGGATCG 552085 8-10-2 83 721 1264 1280 TTCCGCAGTATGGATCG 552141 2-9-6 72 1298 1265 1284 GGAGTTCCGCAGTATGGATC 552086 8-10-2 83 1349 1265 1281 GTTCCGCAGTATGGATC 552142 2-9-6 58 1299 1266 1285 AGGAGTTCCGCAGTATGGAT 552087 8-10-2 77 722 1266 1282 AGTTCCGCAGTATGGAT 552143 2-9-6 70 1300 1267 1283 GAGTTCCGCAGTATGGA 552144 2-9-6 66 1301 1268 1284 GGAGTTCCGCAGTATGG 552145 2-9-6 78 1302 1269 1285 AGGAGTTCCGCAGTATG 552146 2-9-6 63 1303 1577 1596 GTGAAGCGAAGTGCACACGG 552088 8-10-2 90 224 1577 1593 AAGCGAAGTGCACACGG 552147 2-9-6 80 1304 1578 1597 GGTGAAGCGAAGTGCACACG 552089 8-10-2 87 801 1578 1594 GAAGCGAAGTGCACACG 552148 2-9-6 74 1305 1579 1598 AGGTGAAGCGAAGTGCACAC 552090 8-10-2 85 802 1579 1595 TGAAGCGAAGTGCACAC 552149 2-9-6 79 1306 1580 1599 GAGGTGAAGCGAAGTGCACA 552091 8-10-2 84 225 1581 1600 AGAGGTGAAGCGAAGTGCAC 552092 8-10-2 86 804 1582 1601 CAGAGGTGAAGCGAAGTGCA 552093 8-10-2 82 805 1583 1602 GCAGAGGTGAAGCGAAGTGC 552094 8-10-2 84 226 1584 1603 TGCAGAGGTGAAGCGAAGTG 552063 7-10-3 79 806 1584 1603 TGCAGAGGTGAAGCGAAGTG 552095 8-10-2 85 806 1585 1604 GTGCAGAGGTGAAGCGAAGT 552064 7-10-3 83 807 1585 1604 GTGCAGAGGTGAAGCGAAGT 552096 8-10-2 88 807 1586 1605 CGTGCAGAGGTGAAGCGAAG 552065 7-10-3 86 227 1586 1605 CGTGCAGAGGTGAAGCGAAG 552097 8-10-2 90 227 1587 1606 ACGTGCAGAGGTGAAGCGAA 552066 7-10-3 35 1350 1587 1606 ACGTGCAGAGGTGAAGCGAA 552098 8-10-2 86 1350 1778 1797 AATTTATGCCTACAGCCTCC 552067 7-10-3 53 46 1778 1797 AATTTATGCCTACAGCCTCC 552099 8-10-2 66 46 1779 1798 CAATTTATGCCTACAGCCTC 552068 7-10-3 70 48 1779 1798 CAATTTATGCCTACAGCCTC 552100 8-10-2 67 48 1780 1799 CCAATTTATGCCTACAGCCT 552069 7-10-3 68 50 1780 1799 CCAATTTATGCCTACAGCCT 552101 8-10-2 65 50 1781 1800 ACCAATTTATGCCTACAGCC 552070 7-10-3 64 52 1781 1800 ACCAATTTATGCCTACAGCC 552102 8-10-2 54 52

TABLE 46 Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 5-10-5 69 224 57 411 427 GGCATAGCAGCAGGATG 510100 3-10-4 59 17 58 74 CTGGAGCCACCAGCAGG 552330 6-9-2 50 1288 59 75 ACTGGAGCCACCAGCAG 552331 6-9-2 46 1289 60 76 AACTGGAGCCACCAGCA 552332 6-9-2 50 1290 61 77 GAACTGGAGCCACCAGC 552333 6-9-2 48 1291 253 269 GAAGTCCACCACGAGTC 552334 6-9-2 42 9 254 270 AGAAGTCCACCACGAGT 552335 6-9-2 30 10 255 271 GAGAAGTCCACCACGAG 552336 6-9-2 23 11 256 272 AGAGAAGTCCACCACGA 552337 6-9-2 42 12 411 427 GGCATAGCAGCAGGATG 552338 6-9-2 40 17 412 428 AGGCATAGCAGCAGGAT 552339 6-9-2 50 18 413 429 GAGGCATAGCAGCAGGA 552340 6-9-2 45 19 414 430 TGAGGCATAGCAGCAGG 552341 6-9-2 44 21 415 431 ATGAGGCATAGCAGCAG 552342 6-9-2 51 23 416 432 GATGAGGCATAGCAGCA 552343 6-9-2 44 25 417 433 AGATGAGGCATAGCAGC 552344 6-9-2 24 27 418 434 AAGATGAGGCATAGCAG 552345 6-9-2 41 29 457 473 ACGGGCAACATACCTTG 552346 6-9-2 0 33 670 686 ACTAGTAAACTGAGCCA 552347 6-9-2 75 1292 671 687 CACTAGTAAACTGAGCC 552348 6-9-2 72 1293 672 688 GCACTAGTAAACTGAGC 552349 6-9-2 65 1294 687 703 ACCACTGAACAAATGGC 552350 6-9-2 42 40 688 704 AACCACTGAACAAATGG 552351 6-9-2 45 41 689 705 GAACCACTGAACAAATG 552352 6-9-2 43 42 690 706 CGAACCACTGAACAAAT 552353 6-9-2 20 43 1261 1277 CGCAGTATGGATCGGCA 552354 6-9-2 70 1295 1262 1278 CCGCAGTATGGATCGGC 552355 6-9-2 66 1296 1263 1279 TCCGCAGTATGGATCGG 552356 6-9-2 62 1297 1264 1280 TTCCGCAGTATGGATCG 552357 6-9-2 53 1298 1265 1281 GTTCCGCAGTATGGATC 552358 6-9-2 57 1299 1266 1282 AGTTCCGCAGTATGGAT 552359 6-9-2 46 1300 1267 1283 GAGTTCCGCAGTATGGA 552360 6-9-2 45 1301 1268 1284 GGAGTTCCGCAGTATGG 552361 6-9-2 44 1302 1269 1285 AGGAGTTCCGCAGTATG 552308 5-9-3 38 1303 1269 1285 AGGAGTTCCGCAGTATG 552362 6-9-2 51 1303 1577 1593 AAGCGAAGTGCACACGG 552309 5-9-3 76 1304 1577 1593 AAGCGAAGTGCACACGG 552363 6-9-2 73 1304 1578 1594 GAAGCGAAGTGCACACG 552310 5-9-3 58 1305 1578 1594 GAAGCGAAGTGCACACG 552364 6-9-2 66 1305 1579 1595 TGAAGCGAAGTGCACAC 552311 5-9-3 38 1306 1579 1595 TGAAGCGAAGTGCACAC 552365 6-9-2 64 1306 1580 1596 GTGAAGCGAAGTGCACA 552150 2-9-6 68 1307 1580 1596 GTGAAGCGAAGTGCACA 552312 5-9-3 75 1307 1580 1596 GTGAAGCGAAGTGCACA 552366 6-9-2 55 1307 1581 1597 GGTGAAGCGAAGTGCAC 552313 5-9-3 66 1308 1581 1597 GGTGAAGCGAAGTGCAC 552367 6-9-2 67 1308 1582 1598 AGGTGAAGCGAAGTGCA 552314 5-9-3 56 1309 1582 1598 AGGTGAAGCGAAGTGCA 552368 6-9-2 41 1309 1583 1599 GAGGTGAAGCGAAGTGC 552315 5-9-3 46 1310 1583 1599 GAGGTGAAGCGAAGTGC 552369 6-9-2 52 1310 1584 1600 AGAGGTGAAGCGAAGTG 552316 5-9-3 55 1311 1584 1600 AGAGGTGAAGCGAAGTG 552370 6-9-2 35 1311 1585 1601 CAGAGGTGAAGCGAAGT 552317 5-9-3 53 1312 1585 1601 CAGAGGTGAAGCGAAGT 552371 6-9-2 58 1312 1586 1602 GCAGAGGTGAAGCGAAG 552318 5-9-3 59 1313 1586 1602 GCAGAGGTGAAGCGAAG 552372 6-9-2 68 1313 1587 1603 TGCAGAGGTGAAGCGAA 552319 5-9-3 56 1314 1587 1603 TGCAGAGGTGAAGCGAA 552373 6-9-2 63 1314 1588 1604 GTGCAGAGGTGAAGCGA 552320 5-9-3 62 1315 1588 1604 GTGCAGAGGTGAAGCGA 552374 6-9-2 70 1315 1589 1605 CGTGCAGAGGTGAAGCG 552321 5-9-3 63 1316 1589 1605 CGTGCAGAGGTGAAGCG 552375 6-9-2 64 1316 1590 1606 ACGTGCAGAGGTGAAGC 552322 5-9-3 52 1317 1590 1606 ACGTGCAGAGGTGAAGC 552376 6-9-2 58 1317 1778 1794 TTATGCCTACAGCCTCC 552323 5-9-3 45 47 1778 1794 TTATGCCTACAGCCTCC 552377 6-9-2 42 47 1779 1795 TTTATGCCTACAGCCTC 552324 5-9-3 49 49 1779 1795 TTTATGCCTACAGCCTC 552378 6-9-2 37 49 1780 1796 ATTTATGCCTACAGCCT 552325 5-9-3 48 51 1780 1796 ATTTATGCCTACAGCCT 552379 6-9-2 57 51 1781 1797 AATTTATGCCTACAGCC 552326 5-9-3 50 53 1781 1797 AATTTATGCCTACAGCC 552380 6-9-2 48 53 1782 1798 CAATTTATGCCTACAGC 552327 5-9-3 13 54 1782 1798 CAATTTATGCCTACAGC 552381 6-9-2 22 54 1783 1799 CCAATTTATGCCTACAG 552328 5-9-3 9 55 1783 1799 CCAATTTATGCCTACAG 552382 6-9-2 20 55 1784 1800 ACCAATTTATGCCTACA 552329 5-9-3 18 56 1784 1800 ACCAATTTATGCCTACA 552383 6-9-2 18 56

Example 15 Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers

Antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. ISIS 146786 and ISIS 509934, which were described in an earlier application (U.S. Provisional Application No. 61/478,040 filed on Apr. 21, 2011), were also included in these studies for comparison. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000 with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 1; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 2; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 3) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 311; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 312; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 313). HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in Tables below were designed as deoxy, MOE and (S)-cEt gapmers. The gapmers are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in the Tables is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). The potency of the newly designed oligonucleotides was compared with ISIS 146786, 509934, ISIS 509959, and ISIS 510100.

TABLE 47  Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 1780 1799 CCAATTTATGCCTACAGCCT 509934 eeeee-10-eeeee 30 50 58 73 TGGAGCCACCAGCAGG 552787 ekk-10-kke 57 1318 59 74 CTGGAGCCACCAGCAG 552788 ekk-10-kke 60 1319 60 75 ACTGGAGCCACCAGCA 552789 ekk-10-kke 67 1320 61 76 AACTGGAGCCACCAGC 552790 ekk-10-kke 67 1321 62 77 GAACTGGAGCCACCAG 552791 ekk-10-kke 65 86 245 260 CACGAGTCTAGACTCT 552792 ekk-10-kke 44 93 246 261 CCACGAGTCTAGACTC 552793 ekk-10-kke 0 95 250 265 TCCACCACGAGTCTAG 552794 ekk-10-kke 54 98 251 266 GTCCACCACGAGTCTA 552795 ekk-10-kke 55 100 252 267 AGTCCACCACGAGTCT 552796 ekk-10-kke 62 102 253 268 AAGTCCACCACGAGTC 552797 ekk-10-kke 59 104 254 269 GAAGTCCACCACGAGT 552798 ekk-10-kke 59 106 255 270 AGAAGTCCACCACGAG 552799 ekk-10-kke 58 109 256 271 GAGAAGTCCACCACGA 552800 ekk-10-kke 62 112 258 273 GAGAGAAGTCCACCAC 552801 ekk-10-kke 65 115 259 274 TGAGAGAAGTCCACCA 552802 ekk-10-kke 53 117 411 426 GCATAGCAGCAGGATG 552803 ekk-10-kke 67 137 412 427 GGCATAGCAGCAGGAT 552804 ekk-10-kke 75 140 413 428 AGGCATAGCAGCAGGA 552805 ekk-10-kke 72 143 414 429 GAGGCATAGCAGCAGG 552806 ekk-10-kke 64 145 415 430 TGAGGCATAGCAGCAG 552807 ekk-10-kke 68 147 416 431 ATGAGGCATAGCAGCA 552808 ekk-10-kke 65 149 417 432 GATGAGGCATAGCAGC 552809 ekk-10-kke 60 151 418 433 AGATGAGGCATAGCAG 552810 ekk-10-kke 59 153 419 434 AAGATGAGGCATAGCA 552811 ekk-10-kke 64 155 420 435 GAAGATGAGGCATAGC 552812 ekk-10-kke 69 157 421 436 AGAAGATGAGGCATAG 552813 ekk-10-kke 64 159 422 437 AAGAAGATGAGGCATA 552814 ekk-10-kke 62 161 457 472 CGGGCAACATACCTTG 552815 ekk-10-kke 61 167 458 473 ACGGGCAACATACCTT 552816 ekk-10-kke 63 168 639 654 GGCCCACTCCCATAGG 552817 ekk-10-kke 42 176 641 656 GAGGCCCACTCCCATA 552818 ekk-10-kke 44 177 642 657 TGAGGCCCACTCCCAT 552819 ekk-10-kke 56 178 643 658 CTGAGGCCCACTCCCA 552820 ekk-10-kke 59 179 670 685 CTAGTAAACTGAGCCA 552821 ekk-10-kke 76 181 671 686 ACTAGTAAACTGAGCC 552822 ekk-10-kke 77 1322 672 687 CACTAGTAAACTGAGC 552823 ekk-10-kke 73 1323 673 688 GCACTAGTAAACTGAG 552824 ekk-10-kke 73 1324 678 693 AAATGGCACTAGTAAA 552825 ekk-10-kke 51 1364 679 694 CAAATGGCACTAGTAA 552826 ekk-10-kke 55 1365 680 695 ACAAATGGCACTAGTA 552827 ekk-10-kke 67 1366 681 696 AACAAATGGCACTAGT 552828 ekk-10-kke 78 1367 682 697 GAACAAATGGCACTAG 552829 ekk-10-kke 72 1368 683 698 TGAACAAATGGCACTA 552830 ekk-10-kke 71 1369 684 699 CTGAACAAATGGCACT 552831 ekk-10-kke 69 1370 685 700 ACTGAACAAATGGCAC 552832 ekk-10-kke 67 1371 686 701 CACTGAACAAATGGCA 552833 ekk-10-kke 65 1372 687 702 CCACTGAACAAATGGC 552834 ekk-10-kke 78 188 688 703 ACCACTGAACAAATGG 552835 ekk-10-kke 70 190 689 704 AACCACTGAACAAATG 552836 ekk-10-kke 64 191 690 705 GAACCACTGAACAAAT 552837 ekk-10-kke 65 192 691 706 CGAACCACTGAACAAA 552838 ekk-10-kke 64 194 738 753 CCACATCATCCATATA 552839 ekk-10-kke 60 199 739 754 ACCACATCATCCATAT 552840 ekk-10-kke 35 201 1176 1191 CAGCAAACACTTGGCA 552841 ekk-10-kke 62 208 1177 1192 TCAGCAAACACTTGGC 552842 ekk-10-kke 67 209 1261 1276 GCAGTATGGATCGGCA 552843 ekk-10-kke 77 211 1262 1277 CGCAGTATGGATCGGC 552844 ekk-10-kke 81 1325 1263 1278 CCGCAGTATGGATCGG 552845 ekk-10-kke 63 1326 1264 1279 TCCGCAGTATGGATCG 552846 ekk-10-kke 79 1327 1265 1280 TTCCGCAGTATGGATC 552847 ekk-10-kke 47 1328 1266 1281 GTTCCGCAGTATGGAT 552848 ekk-10-kke 69 1329 1267 1282 AGTTCCGCAGTATGGA 552849 ekk-10-kke 59 1330 1268 1283 GAGTTCCGCAGTATGG 552850 ekk-10-kke 83 1331 1269 1284 GGAGTTCCGCAGTATG 552851 ekk-10-kke 90 1332 1270 1285 AGGAGTTCCGCAGTAT 552852 ekk-10-kke 89 1333 1577 1592 AGCGAAGTGCACACGG 552853 ekk-10-kke 83 1334 1578 1593 AAGCGAAGTGCACACG 552854 ekk-10-kke 80 1335 1579 1594 GAAGCGAAGTGCACAC 552855 ekk-10-kke 75 1336 1580 1595 TGAAGCGAAGTGCACA 552856 ekk-10-kke 69 1337 1581 1596 GTGAAGCGAAGTGCAC 552857 ekk-10-kke 68 1338 1582 1597 GGTGAAGCGAAGTGCA 552858 ekk-10-kke 79 1339 1583 1598 AGGTGAAGCGAAGTGC 552859 ekk-10-kke 79 1340 1584 1599 GAGGTGAAGCGAAGTG 552860 ekk-10-kke 71 1341 1585 1600 AGAGGTGAAGCGAAGT 552861 ekk-10-kke 68 1342 1586 1601 CAGAGGTGAAGCGAAG 552862 ekk-10-kke 65 1343 1587 1602 GCAGAGGTGAAGCGAA 552863 ekk-10-kke 70 1344 1588 1603 TGCAGAGGTGAAGCGA 552864 ekk-10-kke 71 1345

TABLE 48  Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 58 73 TGGAGCCACCAGCAGG 552787 ekk-10-kke 53 1318 59 74 CTGGAGCCACCAGCAG 552788 ekk-10-kke 45 1319 60 75 ACTGGAGCCACCAGCA 552789 ekk-10-kke 75 1320 61 76 AACTGGAGCCACCAGC 552790 ekk-10-kke 68 1321 62 77 GAACTGGAGCCACCAG 552791 ekk-10-kke 51 86 245 260 CACGAGTCTAGACTCT 552792 ekk-10-kke 38 93 246 261 CCACGAGTCTAGACTC 552793 ekk-10-kke 0 95 250 265 TCCACCACGAGTCTAG 552794 ekk-10-kke 44 98 251 266 GTCCACCACGAGTCTA 552795 ekk-10-kke 56 100 252 267 AGTCCACCACGAGTCT 552796 ekk-10-kke 45 102 253 268 AAGTCCACCACGAGTC 552797 ekk-10-kke 46 104 254 269 GAAGTCCACCACGAGT 552798 ekk-10-kke 53 106 255 270 AGAAGTCCACCACGAG 552799 ekk-10-kke 48 109 256 271 GAGAAGTCCACCACGA 552800 ekk-10-kke 54 112 258 273 GAGAGAAGTCCACCAC 552801 ekk-10-kke 63 115 259 274 TGAGAGAAGTCCACCA 552802  ekk-10-kke 49 117 411 426 GCATAGCAGCAGGATG 552803 ekk-10-kke 71 137 412 427 GGCATAGCAGCAGGAT 552804 ekk-10-kke 64 140 413 428 AGGCATAGCAGCAGGA 552805 ekk-10-kke 70 143 414 429 GAGGCATAGCAGCAGG 552806 ekk-10-kke 67 145 415 430 TGAGGCATAGCAGCAG 552807 ekk-10-kke 61 147 416 431 ATGAGGCATAGCAGCA 552808 ekk-10-kke 83 149 417 432 GATGAGGCATAGCAGC 552809 ekk-10-kke 59 151 418 433 AGATGAGGCATAGCAG 552810 ekk-10-kke 56 153 419 434 AAGATGAGGCATAGCA 552811 ekk-10-kke 62 155 420 435 GAAGATGAGGCATAGC 552812 ekk-10-kke 66 157 421 436 AGAAGATGAGGCATAG 552813 ekk-10-kke 63 159 422 437 AAGAAGATGAGGCATA 552814 ekk-10-kke 65 161 457 472 CGGGCAACATACCTTG 552815 ekk-10-kke 63 167 458 473 ACGGGCAACATACCTT 552816 ekk-10-kke 88 168 639 654 GGCCCACTCCCATAGG 552817 ekk-10-kke 94 176 641 656 GAGGCCCACTCCCATA 552818 ekk-10-kke 82 177 642 657 TGAGGCCCACTCCCAT 552819 ekk-10-kke 80 178 643 658 CTGAGGCCCACTCCCA 552820 ekk-10-kke 84 179 670 685 CTAGTAAACTGAGCCA 552821 ekk-10-kke 71 181 671 686 ACTAGTAAACTGAGCC 552822 ekk-10-kke 85 1322 672 687 CACTAGTAAACTGAGC 552823 ekk-10-kke 71 1323 673 688 GCACTAGTAAACTGAG 552824 ekk-10-kke 81 1324 678 693 AAATGGCACTAGTAAA 552825 ekk-10-kke 51 1364 679 694 CAAATGGCACTAGTAA 552826 ekk-10-kke 64 1365 680 695 ACAAATGGCACTAGTA 552827 ekk-10-kke 61 1366 681 696 AACAAATGGCACTAGT 552828 ekk-10-kke 76 1367 682 697 GAACAAATGGCACTAG 552829 ekk-10-kke 61 1368 683 698 TGAACAAATGGCACTA 552830 ekk-10-kke 59 1369 684 699 CTGAACAAATGGCACT 552831 ekk-10-kke 58 1370 685 700 ACTGAACAAATGGCAC 552832 ekk-10-kke 64 1371 686 701 CACTGAACAAATGGCA 552833 ekk-10-kke 75 1372 687 702 CCACTGAACAAATGGC 552834 ekk-10-kke 84 188 688 703 ACCACTGAACAAATGG 552835 ekk-10-kke 57 190 689 704 AACCACTGAACAAATG 552836 ekk-10-kke 51 191 690 705 GAACCACTGAACAAAT 552837 ekk-10-kke 53 192 691 706 CGAACCACTGAACAAA 552838 ekk-10-kke 48 194 738 753 CCACATCATCCATATA 552839 ekk-10-kke 50 199 739 754 ACCACATCATCCATAT 552840 ekk-10-kke 54 201 1176 1191 CAGCAAACACTTGGCA 552841 ekk-10-kke 61 208 1177 1192 TCAGCAAACACTTGGC 552842 ekk-10-kke 71 209 1261 1276 GCAGTATGGATCGGCA 552843 ekk-10-kke 75 211 1262 1277 CGCAGTATGGATCGGC 552844 ekk-10-kke 78 1325 1263 1278 CCGCAGTATGGATCGG 552845 ekk-10-kke 52 1326 1264 1279 TCCGCAGTATGGATCG 552846 ekk-10-kke 76 1327 1265 1280 TTCCGCAGTATGGATC 552847 ekk-10-kke 61 1328 1266 1281 GTTCCGCAGTATGGAT 552848 ekk-10-kke 72 1329 1267 1282 AGTTCCGCAGTATGGA 552849 ekk-10-kke 87 1330 1268 1283 GAGTTCCGCAGTATGG 552850 ekk-10-kke 76 1331 1269 1284 GGAGTTCCGCAGTATG 552851 ekk-10-kke 76 1332 1270 1285 AGGAGTTCCGCAGTAT 552852 ekk-10-kke 79 1333 1577 1592 AGCGAAGTGCACACGG 552853 ekk-10-kke 82 1334 1578 1593 AAGCGAAGTGCACACG 552854 ekk-10-kke 85 1335 1579 1594 GAAGCGAAGTGCACAC 552855 ekk-10-kke 78 1336 1580 1595 TGAAGCGAAGTGCACA 552856 ekk-10-kke 77 1337 1581 1596 GTGAAGCGAAGTGCAC 552857 ekk-10-kke 75 1338 1582 1597 GGTGAAGCGAAGTGCA 552858 ekk-10-kke 75 1339 1583 1598 AGGTGAAGCGAAGTGC 552859 ekk-10-kke 79 1340 1584 1599 GAGGTGAAGCGAAGTG 552860 ekk-10-kke 71 1341 1585 1600 AGAGGTGAAGCGAAGT 552861 ekk-10-kke 74 1342 1586 1601 CAGAGGTGAAGCGAAG 552862 ekk-10-kke 66 1343 1587 1602 GCAGAGGTGAAGCGAA 552863 ekk-10-kke 70 1344 1588 1603 TGCAGAGGTGAAGCGA 552864 ekk-10-kke 73 1345

TABLE 49  Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Viral Viral Target Target SEQ Start Stop % ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 eeeee-10-eeeee 60 224 58 73 TGGAGCCACCAGCAGG 552889   ek-10-keke 59 1318 59 74 CTGGAGCCACCAGCAG 552890   ek-10-keke 56 1319 60 75 ACTGGAGCCACCAGCA 552891   ek-10-keke 67 1320 61 76 AACTGGAGCCACCAGC 552892   ek-10-keke 65 1321 62 77 GAACTGGAGCCACCAG 552893   ek-10-keke 68 86 250 265 TCCACCACGAGTCTAG 552894   ek-10-keke 71 98 251 266 GTCCACCACGAGTCTA 552895   ek-10-keke 51 100 252 267 AGTCCACCACGAGTCT 552896   ek-10-keke 51 102 253 268 AAGTCCACCACGAGTC 552897   ek-10-keke 43 104 254 269 GAAGTCCACCACGAGT 552898   ek-10-keke 43 106 255 270 AGAAGTCCACCACGAG 552899   ek-10-keke 55 109 256 271 GAGAAGTCCACCACGA 552900   ek-10-keke 34 112 258 273 GAGAGAAGTCCACCAC 552901   ek-10-keke 42 115 259 274 TGAGAGAAGTCCACCA 552902   ek-10-keke 60 117 411 426 GCATAGCAGCAGGATG 552903   ek-10-keke 76 137 412 427 GGCATAGCAGCAGGAT 552904   ek-10-keke 74 140 413 428 AGGCATAGCAGCAGGA 552905   ek-10-keke 66 143 415 430 TGAGGCATAGCAGCAG 552907   ek-10-keke 69 147 416 431 ATGAGGCATAGCAGCA 552908   ek-10-keke 63 149 417 432 GATGAGGCATAGCAGC 552909   ek-10-keke 70 151 418 433 AGATGAGGCATAGCAG 552910   ek-10-keke 72 153 457 472 CGGGCAACATACCTTG 552911   ek-10-keke 72 167 458 473 ACGGGCAACATACCTT 552912   ek-10-keke 67 168 670 685 CTAGTAAACTGAGCCA 552913   ek-10-keke 74 181 682 697 GAACAAATGGCACTAG 552914   ek-10-keke 75 1368 684 699 CTGAACAAATGGCACT 552915   ek-10-keke 58 1370 686 701 CACTGAACAAATGGCA 552916   ek-10-keke 74 1372 687 702 CCACTGAACAAATGGC 552917   ek-10-keke 76 188 688 703 ACCACTGAACAAATGG 552918   ek-10-keke 75 190 689 704 AACCACTGAACAAATG 552919   ek-10-keke 55 191 690 705 GAACCACTGAACAAAT 552920   ek-10-keke 49 192 691 706 CGAACCACTGAACAAA 552921   ek-10-keke 45 194 1261 1276 GCAGTATGGATCGGCA 552922   ek-10-keke 83 211 1262 1277 CGCAGTATGGATCGGC 552923   ek-10-keke 83 1325 1263 1278 CCGCAGTATGGATCGG 552924   ek-10-keke 0 1326 1264 1279 TCCGCAGTATGGATCG 552925   ek-10-keke 85 1327 1265 1280 TTCCGCAGTATGGATC 552926   ek-10-keke 50 1328 1266 1281 GTTCCGCAGTATGGAT 552927   ek-10-keke 76 1329 1267 1282 AGTTCCGCAGTATGGA 552928   ek-10-keke 78 1330 1268 1283 GAGTTCCGCAGTATGG 552929   ek-10-keke 75 1331 1269 1284 GGAGTTCCGCAGTATG 552930   ek-10-keke 78 1332 1270 1285 AGGAGTTCCGCAGTAT 552931   ek-10-keke 74 1333 1577 1592 AGCGAAGTGCACACGG 552932   ek-10-keke 86 1334 1578 1593 AAGCGAAGTGCACACG 552933   ek-10-keke 82 1335 1579 1594 GAAGCGAAGTGCACAC 552934   ek-10-keke 74 1336 1580 1595 TGAAGCGAAGTGCACA 552935   ek-10-keke 76 1337 1581 1596 GTGAAGCGAAGTGCAC 552936   ek-10-keke 81 1338 1582 1597 GGTGAAGCGAAGTGCA 552937   ek-10-keke 80 1339 1583 1598 AGGTGAAGCGAAGTGC 552938   ek-10-keke 78 1340 1584 1599 GAGGTGAAGCGAAGTG 552939   ek-10-keke 75 1341 1585 1600 AGAGGTGAAGCGAAGT 552940   ek-10-keke 63 1342 1586 1601 CAGAGGTGAAGCGAAG 552941 ekk-10-kke 78 1343 1587 1602 GCAGAGGTGAAGCGAA 552942   ek-10-keke 80 1344 1589 1604 GTGCAGAGGTGAAGCG 552865 ekk-10-kke 67 1346 1590 1605 CGTGCAGAGGTGAAGC 552866 ekk-10-kke 68 1347 1778 1793 TATGCCTACAGCCTCC 552868 ekk-10-kke 55 230 1779 1794 TTATGCCTACAGCCTC 552869 ekk-10-kke 48 231 1780 1795 TTTATGCCTACAGCCT 552870 ekk-10-kke 55 232 1781 1796 ATTTATGCCTACAGCC 552871 ekk-10-kke 57 233 1782 1797 AATTTATGCCTACAGC 552872 ekk-10-kke 70 234 1783 1798 CAATTTATGCCTACAG 552873 ekk-10-kke 49 235 1784 1799 CCAATTTATGCCTACA 552874 ekk-10-kke 42 236 1785 1800 ACCAATTTATGCCTAC 552875 ekk-10-kke 41 237 1822 1837 GGCAGAGGTGAAAAAG 552876 ekk-10-kke 50 244 1823 1838 AGGCAGAGGTGAAAAA 552877   ek-10-keke 39 245 1824 1839 TAGGCAGAGGTGAAAA 552878 ekk-10-kke 31 247 1865 1880 AGCTTGGAGGCTTGAA 552879 ekk-10-kke 5 252 1866 1881 CAGCTTGGAGGCTTGA 552880 ekk-10-kke 5 254 1867 1882 ACAGCTTGGAGGCTTG 552881 ekk-10-kke 10 256 1868 1883 CACAGCTTGGAGGCTT 552882 ekk-10-kke 11 258 1869 1884 GCACAGCTTGGAGGCT 552883 ekk-10-kke 27 260 1870 1885 GGCACAGCTTGGAGGC 552884 ekk-10-kke 36 262 1871 1886 AGGCACAGCTTGGAGG 552885 ekk-10-kke 12 264 1872 1887 AAGGCACAGCTTGGAG 552886 ekk-10-kke 32 266 1874 1889 CCAAGGCACAGCTTGG 552888 ekk-10-kke 1 271

TABLE 50  Inhibition of viral HBV mRNA levels by chimeric antisense  oligonucleotides measured with RTS3371 Viral Viral Target Target SEQ Start Stop % ID Site Site Sequence ISIS No Motif inhibition NO 1577 1596 GTGAAGCGAAGTGCACACGG 146786 eeeee-10-eeeee 59 224 58 73 TGGAGCCACCAGCAGG 552955 eee-10-kkk 60 1318 59 74 CTGGAGCCACCAGCAG 552956 eee-10-kkk 60 1319 60 75 ACTGGAGCCACCAGCA 552957 eee-10-kkk 64 1320 61 76 AACTGGAGCCACCAGC 552958 eee-10-kkk 56 1321 62 77 GAACTGGAGCCACCAG 552959 eee-10-kkk 59 86 250 265 TCCACCACGAGTCTAG 552960 eee-10-kkk 42 98 251 266 GTCCACCACGAGTCTA 552961 eee-10-kkk 41 100 252 267 AGTCCACCACGAGTCT 552962 eee-10-kkk 35 102 253 268 AAGTCCACCACGAGTC 552963 eee-10-kkk 19 104 254 269 GAAGTCCACCACGAGT 552964 eee-10-kkk 34 106 255 270 AGAAGTCCACCACGAG 552965 eee-10-kkk 42 109 256 271 GAGAAGTCCACCACGA 552966 eee-10-kkk 60 112 258 273 GAGAGAAGTCCACCAC 552967 eee-10-kkk 38 115 259 274 TGAGAGAAGTCCACCA 552968 eee-10-kkk 35 117 411 426 GCATAGCAGCAGGATG 552969 eee-10-kkk 67 137 412 427 GGCATAGCAGCAGGAT 552970 eee-10-kkk 56 140 413 428 AGGCATAGCAGCAGGA 552971 eee-10-kkk 69 143 414 429 GAGGCATAGCAGCAGG 552972 eee-10-kkk 75 145 415 430 TGAGGCATAGCAGCAG 552973 eee-10-kkk 59 145 416 431 ATGAGGCATAGCAGCA 552974 eee-10-kkk 71 149 417 432 GATGAGGCATAGCAGC 552975 eee-10-kkk 56 151 418 433 AGATGAGGCATAGCAG 552976 eee-10-kkk 50 153 457 472 CGGGCAACATACCTTG 552977 eee-10-kkk 56 167 458 473 ACGGGCAACATACCTT 552978 eee-10-kkk 43 168 670 685 CTAGTAAACTGAGCCA 552979 eee-10-kkk 71 181 682 697 GAACAAATGGCACTAG 552980 eee-10-kkk 80 1368 684 699 CTGAACAAATGGCACT 552981 eee-10-kkk 64 1370 686 701 CACTGAACAAATGGCA 552982   ek-10-keke 61 1372 687 702 CCACTGAACAAATGGC 552983 eee-10-kkk 77 188 688 703 ACCACTGAACAAATGG 552984 eee-10-kkk 65 190 689 704 AACCACTGAACAAATG 552985 eee-10-kkk 41 191 690 705 GAACCACTGAACAAAT 552986 eee-10-kkk 30 192 691 706 CGAACCACTGAACAAA 552987 eee-10-kkk 41 194 1261 1276 GCAGTATGGATCGGCA 552988 eee-10-kkk 74 211 1262 1277 CGCAGTATGGATCGGC 552989 eee-10-kkk 85 1325 1263 1278 CCGCAGTATGGATCGG 552990 eee-10-kkk 72 1326 1264 1279 TCCGCAGTATGGATCG 552991 eee-10-kkk 73 1327 1265 1280 TTCCGCAGTATGGATC 552992 eee-10-kkk 60 1328 1266 1281 GTTCCGCAGTATGGAT 552993 eee-10-kkk 52 1329 1267 1282 AGTTCCGCAGTATGGA 552994 eee-10-kkk 58 1330 1268 1283 GAGTTCCGCAGTATGG 552995 eee-10-kkk 70 1331 1269 1284 GGAGTTCCGCAGTATG 552996 eee-10-kkk 74 1332 1270 1285 AGGAGTTCCGCAGTAT 552997 eee-10-kkk 59 1333 1577 1592 AGCGAAGTGCACACGG 552998 eee-10-kkk 82 1334 1578 1593 AAGCGAAGTGCACACG 552999 eee-10-kkk 70 1335 1579 1594 GAAGCGAAGTGCACAC 553000 eee-10-kkk 67 1336 1580 1595 TGAAGCGAAGTGCACA 553001 eee-10-kkk 67 1337 1581 1596 GTGAAGCGAAGTGCAC 553002 eee-10-kkk 74 1338 1582 1597 GGTGAAGCGAAGTGCA 553003 eee-10-kkk 72 1339 1583 1598 AGGTGAAGCGAAGTGC 553004 eee-10-kkk 73 1340 1584 1599 GAGGTGAAGCGAAGTG 553005 eee-10-kkk 67 1341 1585 1600 AGAGGTGAAGCGAAGT 553006 eee-10-kkk 69 1342 1586 1601 CAGAGGTGAAGCGAAG 553007 eee-10-kkk 60 1343 1587 1602 GCAGAGGTGAAGCGAA 553008 eee-10-kkk 71 1344 1588 1603 TGCAGAGGTGAAGCGA 552943   ek-10-keke 77 1345 1588 1603 TGCAGAGGTGAAGCGA 553009 eee-10-kkk 78 1345 1589 1604 GTGCAGAGGTGAAGCG 552944   ek-10-keke 74 1346 1589 1604 GTGCAGAGGTGAAGCG 553010 eee-10-kkk 78 1346 1590 1605 CGTGCAGAGGTGAAGC 552945   ek-10-keke 76 1347 1590 1605 CGTGCAGAGGTGAAGC 553011 eee-10-kkk 72 1347 1591 1606 ACGTGCAGAGGTGAAG 552946   ek-10-keke 71 1348 1591 1606 ACGTGCAGAGGTGAAG 553012 eee-10-kkk 74 1348 1778 1793 TATGCCTACAGCCTCC 552947   ek-10-keke 54 230 1778 1793 TATGCCTACAGCCTCC 553013 eee-10-kkk 39 230 1779 1794 TTATGCCTACAGCCTC 552948   ek-10-keke 50 231 1779 1794 TTATGCCTACAGCCTC 553014 eee-10-kkk 37 231 1780 1795 TTTATGCCTACAGCCT 552949   ek-10-keke 8 232 1780 1795 TTTATGCCTACAGCCT 553015 eee-10-kkk 45 232 1781 1796 ATTTATGCCTACAGCC 552950   ek-10-keke 44 233 1781 1796 ATTTATGCCTACAGCC 553016 eee-10-kkk 47 233 1782 1797 AATTTATGCCTACAGC 552951   ek-10-keke 60 234 1782 1797 AATTTATGCCTACAGC 553017 eee-10-kkk 47 234 1783 1798 CAATTTATGCCTACAG 552952   ek-10-keke 35 235 1783 1798 CAATTTATGCCTACAG 553018 eee-10-kkk 30 235 1784 1799 CCAATTTATGCCTACA 552953   ek-10-keke 37 236 1784 1799 CCAATTTATGCCTACA 553019 eee-10-kkk 37 236 1785 1800 ACCAATTTATGCCTAC 552954   ek-10-keke 40 237 1785 1800 ACCAATTTATGCCTAC 553020 eee-10-kkk 24 237

TABLE 51  Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 58 73 TGGAGCCACCAGCAGG 552889 ek-10-keke 42 1318 59 74 CTGGAGCCACCAGCAG 552890 ek-10-keke 56 1319 60 75 ACTGGAGCCACCAGCA 552891 ek-10-keke 55 1320 61 76 AACTGGAGCCACCAGC 552892 ek-10-keke 53 1321 62 77 GAACTGGAGCCACCAG 552893 ek-10-keke 56 86 250 265 TCCACCACGAGTCTAG 552894 ek-10-keke 53 98 251 266 GTCCACCACGAGTCTA 552895 ek-10-keke 38 100 252 267 AGTCCACCACGAGTCT 552896 ek-10-keke 43 102 253 268 AAGTCCACCACGAGTC 552897 ek-10-keke 40 104 254 269 GAAGTCCACCACGAGT 552898 ek-10-keke 50 106 255 270 AGAAGTCCACCACGAG 552899 ek-10-keke 37 109 256 271 GAGAAGTCCACCACGA 552900 ek-10-keke 43 112 258 273 GAGAGAAGTCCACCAC 552901 ek-10-keke 56 115 259 274 TGAGAGAAGTCCACCA 552902 ek-10-keke 43 117 411 426 GCATAGCAGCAGGATG 552903 ek-10-keke 78 137 412 427 GGCATAGCAGCAGGAT 552904 ek-10-keke 75 140 413 428 AGGCATAGCAGCAGGA 552905 ek-10-keke 52 143 415 430 TGAGGCATAGCAGCAG 552907 ek-10-keke 75 147 416 431 ATGAGGCATAGCAGCA 552908 ek-10-keke 57 149 417 432 GATGAGGCATAGCAGC 552909 ek-10-keke 66 151 418 433 AGATGAGGCATAGCAG 552910 ek-10-keke 60 153 457 472 CGGGCAACATACCTTG 552911 ek-10-keke 65 167 458 473 ACGGGCAACATACCTT 552912 ek-10-keke 37 168 670 685 CTAGTAAACTGAGCCA 552913 ek-10-keke 76 181 682 697 GAACAAATGGCACTAG 552914 ek-10-keke 79 1368 684 699 CTGAACAAATGGCACT 552915 ek-10-keke 71 1370 686 701 CACTGAACAAATGGCA 552916 ek-10-keke 82 1372 687 702 CCACTGAACAAATGGC 552917 ek-10-keke 78 188 688 703 ACCACTGAACAAATGG 552918 ek-10-keke 64 190 689 704 AACCACTGAACAAATG 552919 ek-10-keke 38 191 690 705 GAACCACTGAACAAAT 552920 ek-10-keke 43 192 691 706 CGAACCACTGAACAAA 552921 ek-10-keke 49 194 1261 1276 GCAGTATGGATCGGCA 552922 ek-10-keke 90 211 1262 1277 CGCAGTATGGATCGGC 552923 ek-10-keke 92 1325 1263 1278 CCGCAGTATGGATCGG 552924 ek-10-keke 30 1326 1264 1279 TCCGCAGTATGGATCG 552925 ek-10-keke 81 1327 1265 1280 TTCCGCAGTATGGATC 552926 ek-10-keke 39 1328 1266 1281 GTTCCGCAGTATGGAT 552927 ek-10-keke 53 1329 1267 1282 AGTTCCGCAGTATGGA 552928 ek-10-keke 48 1330 1268 1283 GAGTTCCGCAGTATGG 552929 ek-10-keke 68 1331 1269 1284 GGAGTTCCGCAGTATG 552930 ek-10-keke 87 1332 1270 1285 AGGAGTTCCGCAGTAT 552931 ek-10-keke 87 1333 1577 1592 AGCGAAGTGCACACGG 552932 ek-10-keke 88 1334 1578 1593 AAGCGAAGTGCACACG 552933 ek-10-keke 75 1335 1579 1594 GAAGCGAAGTGCACAC 552934 ek-10-keke 76 1336 1580 1595 TGAAGCGAAGTGCACA 552935 ek-10-keke 71 1337 1581 1596 GTGAAGCGAAGTGCAC 552936 ek-10-keke 80 1338 1582 1597 GGTGAAGCGAAGTGCA 552937 ek-10-keke 81 1339 1583 1598 AGGTGAAGCGAAGTGC 552938 ek-10-keke 85 1340 1584 1599 GAGGTGAAGCGAAGTG 552939 ek-10-keke 82 1341 1585 1600 AGAGGTGAAGCGAAGT 552940 ek-10-keke 76 1342 1586 1601 CAGAGGTGAAGCGAAG 552941 ekk-10-kke 72 1343 1587 1602 GCAGAGGTGAAGCGAA 552942 ek-10-keke 85 1344 1589 1604 GTGCAGAGGTGAAGCG 552865 ekk-10-kke 70 1346 1590 1605 CGTGCAGAGGTGAAGC 552866 ekk-10-kke 65 1347 1778 1793 TATGCCTACAGCCTCC 552868 ekk-10-kke 36 230 1779 1794 TTATGCCTACAGCCTC 552869 ekk-10-kke 23 231 1780 1795 TTTATGCCTACAGCCT 552870 ekk-10-kke 49 232 1781 1796 ATTTATGCCTACAGCC 552871 ekk-10-kke 46 233 1782 1797 AATTTATGCCTACAGC 552872 ekk-10-kke 73 234 1783 1798 CAATTTATGCCTACAG 552873 ekk-10-kke 41 235 1784 1799 CCAATTTATGCCTACA 552874 ekk-10-kke 18 236 1785 1800 ACCAATTTATGCCTAC 552875 ekk-10-kke 0 237 1822 1837 GGCAGAGGTGAAAAAG 552876 ekk-10-kke 49 244 1823 1838 AGGCAGAGGTGAAAAA 552877 ek-10-keke 37 245 1824 1839 TAGGCAGAGGTGAAAA 552878 ekk-10-kke 28 247 1865 1880 AGCTTGGAGGCTTGAA 552879 ekk-10-kke 0 252 1866 1881 CAGCTTGGAGGCTTGA 552880 ekk-10-kke 12 254 1867 1882 ACAGCTTGGAGGCTTG 552881 ekk-10-kke 0 256 1868 1883 CACAGCTTGGAGGCTT 552882 ekk-10-kke 0 258 1869 1884 GCACAGCTTGGAGGCT 552883 ekk-10-kke 12 260 1870 1885 GGCACAGCTTGGAGGC 552884 ekk-10-kke 39 262 1871 1886 AGGCACAGCTTGGAGG 552885 ekk-10-kke 37 264 1872 1887 AAGGCACAGCTTGGAG 552886 ekk-10-kke 15 266 1874 1889 CCAAGGCACAGCTTGG 552888 ekk-10-kke 0 271

TABLE 52  Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Viral Viral Target Target Start Stop % SEQ ID Site Site Sequence ISIS No Motif inhibition NO 58 73 TGGAGCCACCAGCAGG 552955 eee-10-kkk 67 1318 59 74 CTGGAGCCACCAGCAG 552956 eee-10-kkk 60 1319 60 75 ACTGGAGCCACCAGCA 552957 eee-10-kkk 73 1320 61 76 AACTGGAGCCACCAGC 552958 eee-10-kkk 63 1321 62 77 GAACTGGAGCCACCAG 552959 eee-10-kkk 58 86 250 265 TCCACCACGAGTCTAG 552960 eee-10-kkk 67 98 251 266 GTCCACCACGAGTCTA 552961 eee-10-kkk 78 100 252 267 AGTCCACCACGAGTCT 552962 eee-10-kkk 29 102 253 268 AAGTCCACCACGAGTC 552963 eee-10-kkk 25 104 254 269 GAAGTCCACCACGAGT 552964 eee-10-kkk 33 106 255 270 AGAAGTCCACCACGAG 552965 eee-10-kkk 55 109 256 271 GAGAAGTCCACCACGA 552966 eee-10-kkk 71 112 258 273 GAGAGAAGTCCACCAC 552967 eee-10-kkk 23 115 259 274 TGAGAGAAGTCCACCA 552968 eee-10-kkk 41 117 411 426 GCATAGCAGCAGGATG 552969 eee-10-kkk 76 137 412 427 GGCATAGCAGCAGGAT 552970 eee-10-kkk 44 140 413 428 AGGCATAGCAGCAGGA 552971 eee-10-kkk 77 143 414 429 GAGGCATAGCAGCAGG 552972 eee-10-kkk 74 145 415 430 TGAGGCATAGCAGCAG 552973 eee-10-kkk 61 145 416 431 ATGAGGCATAGCAGCA 552974 eee-10-kkk 73 149 417 432 GATGAGGCATAGCAGC 552975 eee-10-kkk 66 151 418 433 AGATGAGGCATAGCAG 552976 eee-10-kkk 70 153 457 472 CGGGCAACATACCTTG 552977 eee-10-kkk 65 167 458 473 ACGGGCAACATACCTT 552978 eee-10-kkk 40 168 670 685 CTAGTAAACTGAGCCA 552979 eee-10-kkk 79 181 682 697 GAACAAATGGCACTAG 552980 eee-10-kkk 81 64 684 699 CTGAACAAATGGCACT 552981 eee-10-kkk 74 66 686 701 CACTGAACAAATGGCA 552982   ek-10-keke 52 68 687 702 CCACTGAACAAATGGC 552983 eee-10-kkk 78 188 688 703 ACCACTGAACAAATGG 552984 eee-10-kkk 71 190 689 704 AACCACTGAACAAATG 552985 eee-10-kkk 38 191 690 705 GAACCACTGAACAAAT 552986 eee-10-kkk 48 192 691 706 CGAACCACTGAACAAA 552987 eee-10-kkk 54 194 1261 1276 GCAGTATGGATCGGCA 552988 eee-10-kkk 85 211 1262 1277 CGCAGTATGGATCGGC 552989 eee-10-kkk 84 1325 1263 1278 CCGCAGTATGGATCGG 552990 eee-10-kkk 79 1326 1264 1279 TCCGCAGTATGGATCG 552991 eee-10-kkk 53 1327 1265 1280 TTCCGCAGTATGGATC 552992 eee-10-kkk 68 1328 1266 1281 GTTCCGCAGTATGGAT 552993 eee-10-kkk 67 1329 1267 1282 AGTTCCGCAGTATGGA 552994 eee-10-kkk 69 1330 1268 1283 GAGTTCCGCAGTATGG 552995 eee-10-kkk 62 1331 1269 1284 GGAGTTCCGCAGTATG 552996 eee-10-kkk 82 1332 1270 1285 AGGAGTTCCGCAGTAT 552997 eee-10-kkk 58 1333 1577 1592 AGCGAAGTGCACACGG 552998 eee-10-kkk 86 1334 1578 1593 AAGCGAAGTGCACACG 552999 eee-10-kkk 63 1335 1579 1594 GAAGCGAAGTGCACAC 553000 eee-10-kkk 67 1336 1580 1595 TGAAGCGAAGTGCACA 553001 eee-10-kkk 70 1337 1581 1596 GTGAAGCGAAGTGCAC 553002 eee-10-kkk 84 1338 1582 1597 GGTGAAGCGAAGTGCA 553003 eee-10-kkk 83 1339 1583 1598 AGGTGAAGCGAAGTGC 553004 eee-10-kkk 68 1340 1584 1599 GAGGTGAAGCGAAGTG 553005 eee-10-kkk 57 1341 1585 1600 AGAGGTGAAGCGAAGT 553006 eee-10-kkk 74 1342 1586 1601 CAGAGGTGAAGCGAAG 553007 eee-10-kkk 62 1343 1587 1602 GCAGAGGTGAAGCGAA 553008 eee-10-kkk 50 1344 1588 1603 TGCAGAGGTGAAGCGA 552943   ek-10-keke 86 1345 1588 1603 TGCAGAGGTGAAGCGA 553009 eee-10-kkk 79 1345 1589 1604 GTGCAGAGGTGAAGCG 552944   ek-10-keke 83 1346 1589 1604 GTGCAGAGGTGAAGCG 553010 eee-10-kkk 74 1346 1590 1605 CGTGCAGAGGTGAAGC 552945   ek-10-keke 79 1347 1590 1605 CGTGCAGAGGTGAAGC 553011 eee-10-kkk 60 1347 1591 1606 ACGTGCAGAGGTGAAG 552946   ek-10-keke 68 1348 1591 1606 ACGTGCAGAGGTGAAG 553012 eee-10-kkk 78 1348 1778 1793 TATGCCTACAGCCTCC 552947   ek-10-keke 51 230 1778 1793 TATGCCTACAGCCTCC 553013 eee-10-kkk 45 230 1779 1794 TTATGCCTACAGCCTC 552948   ek-10-keke 56 231 1779 1794 TTATGCCTACAGCCTC 553014 eee-10-kkk 53 231 1780 1795 TTTATGCCTACAGCCT 552949   ek-10-keke 1 232 1780 1795 TTTATGCCTACAGCCT 553015 eee-10-kkk 55 232 1781 1796 ATTTATGCCTACAGCC 552950   ek-10-keke 52 233 1781 1796 ATTTATGCCTACAGCC 553016 eee-10-kkk 65 233 1782 1797 AATTTATGCCTACAGC 552951   ek-10-keke 59 234 1782 1797 AATTTATGCCTACAGC 553017 eee-10-kkk 36 234 1783 1798 CAATTTATGCCTACAG 552952   ek-10-keke 34 235 1783 1798 CAATTTATGCCTACAG 553018 eee-10-kkk 20 235 1784 1799 CCAATTTATGCCTACA 552953   ek-10-keke 55 236 1784 1799 CCAATTTATGCCTACA 553019 eee-10-kkk 34 236 1785 1800 ACCAATTTATGCCTAC 552954   ek-10-keke 51 237 1785 1800 ACCAATTTATGCCTAC 553020 eee-10-kkk 28 237

Example 16 Dose-Dependent Antisense Inhibition of HBV mRNA in HepG2 Cells by MOE Gapmers

Antisense oligonucleotides from the study described in Example 14 exhibiting in vitro inhibition of HBV mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000 with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 53. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. HBV primer probe set RTS3371 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

As illustrated in Table 53, HBV mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. ‘n/a’ indicates that the data for that dosage is not available.

TABLE 53 Dose-dependent antisense inhibition of human HBV in HepG2 cells ISIS No 9.2593 nM 27.7778 nM 83.3333 nM 250.0 nM 146786 10 43 74 89 509934 12 31 52 79 509959 4 24 49 67 510100 11 28 60 77 510124 3 11 13 41 551926 1 26 51 76 551958 15 17 56 82 551987 4 40 65 81 551990 7 55 78 91 551993 15 30 70 80 551994 0 30 39 58 551995 6 41 73 85 551996 13 47 71 85 551997 16 38 68 89 551998 4 36 69 85 551999 10 31 67 86 552000 0 17 61 78 552006 6 37 74 89 552009 1 5 39 60 552013 0 28 3 72 552014 0 26 32 77 552018 6 27 63 81 552019 15 34 65 90 552020 2 35 65 91 552021 4 11 53 82 552022 6 35 57 79 552023 11 33 59 81 552024 15 43 69 91 552025 17 35 69 87 552026 14 26 66 86 552027 3 46 62 88 552028 9 43 58 78 552029 8 40 72 89 552030 18 48 77 92 552031 0 38 66 89 552032 42 48 80 88 552033 2 40 64 84 552034 6 40 70 81 552039 2 33 56 83 552044 19 30 63 84 552046 4 21 47 77 552050 15 44 70 92 552051 8 33 69 90 552052 17 38 71 91 552053 0 40 59 86 552054 7 15 58 75 552056 19 62 86 92 552057 11 33 69 86 552058 30 55 79 90 552059 11 25 69 90 552060 9 32 61 86 552061 6 40 69 88 552062 22 48 75 89 552064 23 49 69 90 552065 10 8 69 86 552069 11 4 28 60 552073 9 31 62 78 552075 21 18 33 65 552077 0 17 40 72 552079 1 12 44 70 552080 3 12 34 69 552082 13 29 66 87 552083 24 54 69 88 552084 10 25 48 82 552085 28 35 64 85 552086 0 24 65 84 552088 33 53 77 93 552089 0 41 69 92 552090 17 35 70 87 552091 13 31 69 89 552092 6 23 66 89 552093 0 17 61 89 552094 12 38 65 88 552095 20 42 73 88 552096 n/a 39 66 91 552097 24 43 67 88 552098 0 24 56 85 552101 3 13 28 61 552147 11 27 58 80 552160 20 25 69 89 552163 0 21 22 53 552176 16 11 40 66 552192 7 38 78 89 552222 0 24 65 79 552247 0 38 69 86 552255 5 27 69 81 552301 5 38 65 86 552309 8 26 62 85 552312 0 4 32 62 552347 2 15 38 75 552348 12 40 42 65 552354 10 35 44 76 552361 2 25 55 74 552363 20 36 54 76 552374 7 4 38 76 552379 0 12 24 46 552403 8 27 54 76 552408 2 25 44 77 552409 6 31 56 80 552418 0 30 72 84 552420 9 34 53 81 552442 4 23 46 56 552466 0 23 56 79 552474 11 34 66 87 552477 11 22 44 64 552530 25 37 73 87 552559 9 13 29 51

Example 17 Dose-Dependent Antisense Inhibition of HBV mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers

Antisense oligonucleotides from the study described in Example 15 exhibiting in vitro inhibition of HBV mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000 with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 54. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. HBV primer probe set RTS3371 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

As illustrated in Table 54, HBV mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 54 Dose-dependent antisense inhibition of human HBV in HepG2 cells ISIS No 9.2593 nM 27.7778 nM 83.3333 nM 250.0 nM 146786 10 43 74 89 552808 13 14 55 70 552816 38 73 87 92 552818 29 63 87 85 552820 58 83 90 90 552821 33 49 71 88 552822 24 55 74 88 552824 8 24 65 87 552834 11 28 68 89 552849 12 25 73 84 552851 13 42 74 89 552852 4 35 70 87 552853 19 52 86 93 552854 28 57 80 89 552916 5 26 64 82 552922 25 44 77 89 552923 22 49 82 91 552925 33 56 80 92 552930 12 49 79 89 552931 12 40 62 82 552932 24 62 84 91 552933 20 40 75 89 552936 18 36 75 88 552937 22 51 82 88 552938 12 36 67 80 552939 17 40 65 79 552942 21 48 74 88 552943 5 39 70 85 552944 14 33 70 77 552980 15 40 69 86 552988 4 36 58 84 552989 0 50 74 81 552996 0 25 53 72 552998 17 49 79 90 553002 0 32 68 86 553003 15 42 67 88

Example 18 Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. ISIS 5808 and ISIS 9591, disclosed in U.S. Pat. No. 5,985,662, as well as ISIS 146781, ISIS 146786, 524518, ISIS 552859, and ISIS 552870 were also included in these studies for comparison and are distinguished with an asterisk. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000 R with 100 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe sets RTS3370 and RTS3371 and were used to separately measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in Table below were designed as MOE gapmers or deoxy, MOE and (S)-cEt gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleosides each. The deoxy, MOE and (S)-cEt gapmers are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 55 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).

TABLE 55  Inhibition of viral HBV mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 or RTS3371 Viral Viral Target Target % % SEQ Start Stop inhibition inhibition ID Site Site ISIS No Motif (RTS3370) (RTS3371) Sequence NO 156 176   5808* Uniform deoxy 57 64 CCTGATGTGATGTTCTCCATG 1373 303 322 524518* eeeee-10-eeeee 62 72 GGGACTGCGAATTTTGGCCA 428 376 395 146781* eeeee-10-eeeee 72 93 AAACGCCGCAGACACATCCA 1374 380 399 582665  eeeee-10-eeeee 57 59 GATAAAACGCCGCAGACACA 1375 382 401 582666  eeeee-10-eeeee 49 92 ATGATAAAACGCCGCAGACA 1376 411 426 566831  kdkdk-9-ee 96 73 GCATAGCAGCAGGATG 137 411 427 577123   eekk-9-ekee 84 96 GGCATAGCAGCAGGATG 17 411 427 577124  kdkdk-8-eeee 92 96 GGCATAGCAGCAGGATG 17 411 426 577126    kkk-8-eeeee 87 90 GCATAGCAGCAGGATG 137 413 428 566830  kdkdk-9-ee  93 95 AGGCATAGCAGCAGGA 143 415 430 577130    eek-10-kke 87 94 TGAGGCATAGCAGCAG 147 415 430 577131  kdkdk-9-ee 83 93 TGAGGCATAGCAGCAG 147 1263 1278 566828  kdkdk-9-ee 97 90 CCGCAGTATGGATCGG 1236 1577 1596 146786* eeeee-10-eeeee 93 71 GTGAAGCGAAGTGCACACGG 224 1577 1592 566829  kdkdk-9-ee 98 84 AGCGAAGTGCACACGG 1334 1577 1596 577120  kdkdk-10-eeeee 94 93 GTGAAGCGAAGTGCACACGG 224 1577 1592 577127    kkk-8-eeeee 95 70 AGCGAAGTGCACACGG 1334 1577 1592 577134    kek-8-eeeee 94 89 AGCGAAGTGCACACGG 1334 1577 1592 577135    kek-10-kek 96 94 AGCGAAGTGCACACGG 1334 1583 1598 552859*   ekk-10-kke 92 91 AGGTGAAGCGAAGTGC 1340 1583 1602 577121  kdkdk-10-eeeee 91 74 GCAGAGGTGAAGCGAAGTGC 226 1583 1598 577128    kkk-8-eeeee 92 85 AGGTGAAGCGAAGTGC 1340 1583 1598 577132  kdkdk-9-ee  97 81 AGGTGAAGCGAAGTGC 1340 1583 1598 577136    kek-10-kek 95 95 AGGTGAAGCGAAGTGC 1340 1588 1603 566832  kdkdk-9-ee 95 78 TGCAGAGGTGAAGCGA 1345 1780 1795 552870*   ekk-10-kke 71 93 TTTATGCCTACAGCCT 232 1780 1799 577122  kdkdk-10-eeeee 70 96 CCAATTTATGCCTACAGCCT 50 1780 1796 577125  kdkdk-8-eeee 70 94 ATTTATGCCTACAGCCT 51 1780 1795 577129    kkk-8-eeeee 76 51 TTTATGCCTACAGCCT 232 1780 1795 577133  kdkdk-9-ee 80 52 TTTATGCCTACAGCCT 232 1873 1892   9591* Uniform deoxy 30 14 CACCCAAGGCACAGCTTGG 1377

Example 19 Efficacy of Gapmers Targeting HBV in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides in a number of studies to evaluate the efficacy of the gapmers. HBV DNA and RNA levels were assessed.

Study 1

Groups of 12 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 510106, ISIS 510116, ISIS 505347, or ISIS 509934. A control group of 12 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3370, RTS3371, and RTS3372. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 56, expressed as percent inhibition compared to the control group. As shown in Table 56, most of the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.

TABLE 56 Percent inhibition of HBV RNA and DNA in the liver of transgenic mice % % % % % % inhibition inhibition inhibition inhibition inhibition inhibition ISIS DNA DNA DNA RNA RNA RNA No Chemistry (RTS3370) (RTS3371) (RTS3372) (RTS3370) (RTS3371) (RTS3372) 505347 5-10-5 MOE 72 79 75 54 28 30 509934 5-10-5 MOE 93 95 94 72 75 92 510106 3-10-4 MOE 0 0 51 0 0 12 510116 3-10-4 MOE 68 79 68 49 54 66 Study 2

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 146779, ISIS 505358, ISIS 146786, ISIS 509974, ISIS 509958, or ISIS 509959. A control group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3370. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3370 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 57, expressed as percent inhibition compared to the control group. As shown in Table 57, most of the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.

TABLE 57 Percent inhibition of HBV RNA and DNA in the liver of transgenic mice % % inhibition inhibition ISIS No Chemistry DNA RNA 146779 5-10-5 MOE 39 5 146786 5-10-5 MOE 83 73 505358 5-10-5 MOE 84 77 509958 3-10-3 MOE 82 29 509959 3-10-3 MOE 54 30 509974 3-10-3 MOE 56 28 Study 3

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 509960, ISIS 505329, ISIS 146786, ISIS 505339, or ISIS 509927. Another group of 6 mice was administered Entecavir, an oral antiviral drug used to treated Hepatitis B infection, at 1 mg/kg daily for two weeks. A control group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®.

The data is presented in Table 58, expressed as percent inhibition compared to the control group. As shown in Table 58, most of the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.

TABLE 58 Percent inhibition of HBV RNA and DNA in the liver of transgenic mice % % inhibition inhibition Oligo Chemistry DNA RNA entecavir — 94 0 ISIS 146786 5-10-5 MOE 97 92 ISIS 505329 5-10-5 MOE 70 63 ISIS 505339 5-10-5 MOE 74 63 ISIS 509927 5-10-5 MOE 80 57 ISIS 509960 3-10-3 MOE 86 60 Study 4

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552176, and ISIS 552073. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 59. As shown in Table 59, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.

TABLE 59 Percent inhibition of HBV RNA and DNA in transgenic mice % inhibition of % inhibition of ISIS No Chemistry RNA DNA 146786 5-10-5 MOE 81 91 552073 8-10-2 MOE 39 22 552176 3-9-5 MOE 55 56 Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) (Nyblom, H. et al., Alcohol & Alcoholism 39: 336-339, 2004; Tietz N W (Ed): Clinical Guide to Laboratory Tests, 3rd ed. W. B. Saunders, Philadelphia, Pa., 1995). The results are presented in Table 60 expressed in IU/L. Both the ISIS oligonucleotides were considered tolerable in the mice, as demonstrated by their liver transaminase profile.

TABLE 60 ALT levels (IU/L) of transgenic mice ALT PBS 77 ISIS 146786 21 ISIS 552073 19 ISIS 552176 27 Study 5

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552056, ISIS 552088, and ISIS 552309. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 61, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.

TABLE 61 Percent inhibition of HBV DNA and RNA in transgenic mice % % inhibition inhibition Chemistry (RNA) (DNA) ISIS 146786 5-10-5 MOE 60 90 ISIS 552056 7-10-3 MOE 25 58 ISIS 552088 8-10-2 MOE 8 0 ISIS 552309 5-9-3 MOE 35 84 Study 6

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 505330, ISIS 509932, ISIS 552032, ISIS 552057, ISIS 552075, ISIS 552092, and ISIS 552255. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 62, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column indicates the gap-wing motif of each gapmer.

TABLE 62 Percent inhibition of HBV DNA and RNA in transgenic mice % % inhibition inhibition ISIS No Chemistry (RNA) (DNA) 146786 5-10-5 MOE 52 95 505330 5-10-5 MOE 7 61 509932 5-10-5 MOE 83 98 552032 6-10-4 MOE 54 97 552057 7-10-3 MOE 19 62 552075 8-10-2 MOE 12 18 552092 8-10-2 MOE 25 74 552255 4-9-4 MOE 41 89 Study 7

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 20 mg/kg of ISIS 552859, ISIS 577121, ISIS 577122, ISIS 577123, ISIS 577132, ISIS 577133, and ISIS 577134. These gapmers have deoxy, MOE and (S)-cEt chemistry. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 63, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification.

TABLE 63 Percent inhibition of HBV DNA and RNA in transgenic mice % % inhibition inhibition ISIS No Chemistry (RNA) (DNA) 552859 ekk-10-kke 60 86 577121 kdkdk-10-eeeee 59 93 577122 kdkdk-10-eeeee 42 68 577123 eekk-9-ekee 0 77 577132 kdkdk-9-ee 4 24 577133 kdkdk-9-ee 46 64 577134 kek-8-eeeee 0 17 Study 8

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, the 5-10-5 MOE gapmer. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552803, ISIS 552903, ISIS 552817, ISIS 552822, and ISIS 552907. These gapmers all had deoxy, MOE, and (S)-cEt chemistry. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 64. As shown in Table 64, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification; in case of the MOE gapmers, the Chemistry column defines the gap-wing structure.

TABLE 64 Percent inhibition of HBV RNA and DNA in transgenic mice % % Dose inhibition inhibition ISIS No Chemistry (mg/kg/wk) of RNA of DNA 146786 5-10-5 MOE 50 81 91 552803 ekk-10-kke 20 71 95 552817 ekk-10-kke 20 86 51 552822 ekk-10-kke 20 90 89 552903 ek-10-keke 20 56 82 552907 ek-10-keke 20 41 45 Study 9

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552853, ISIS 552854, ISIS 552932, and ISIS 552938. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 65, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification; in case of the MOE gapmers, the Chemistry column defines the gap-wing structure.

TABLE 65 Percent inhibition of HBV DNA and RNA in transgenic mice % % Dose inhibition inhibition Chemistry (mg/kg/wk) (DNA) (RNA) ISIS 146786 5-10-5 MOE 50 90 60 ISIS 552853 ekk-10-kke 20 94 60 ISIS 552854 ekk-10-kke 20 61 23 ISIS 552932 ek-10-keke 20 75 70 ISIS 552938 ek-10-keke 20 67 56 Study 10

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552922, ISIS 552923, ISIS 552942, ISIS 552872, ISIS 552925, ISIS 552937, and ISIS 552939. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 66, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification; in case of the MOE gapmers, the Chemistry column defines the gap-wing structure.

TABLE 66 Percent inhibition of HBV DNA and RNA in transgenic mice % % Dose inhibition inhibition ISIS No Chemistry (mg/kg/wk) (DNA) (RNA) 146786 5-10-5 MOE 50 52 57 552922 ekk-10-kke 20 61 50 552923 ek-10-keke 20 89 76 552942 ek-10-keke 20 58 52 552872 ek-10-keke 20 77 46 552925 ek-10-keke 20 89 65 552937 ek-10-keke 20 59 35 552939 ek-10-keke 20 57 19

Example 20 Efficacy of Gapmers Targeting HBV in Transgenic Mice

Mice harboring a HBV gene fragment (Guidotti, L. G. et al., J. Virol. 1995, 69, 6158-6169) were used. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model. HBV DNA, RNA, and antigen levels were assessed.

Groups of 10 mice each were injected subcutaneously twice a week for the first with 50 mg/kg and, subsequently, twice a week for the next 3 weeks with 25 mg/kg of ISIS 146786 or ISIS 510100. Control groups of 10 mice each were treated in a similar manner with ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, SEQ ID NO: 320; 5-10-5 MOE gapmer with no known murine target) or ISIS 459024 (CGGTCCTTGGAGGATGC, SEQ ID NO: 1351; 3-10-4 MOE gapmer with no known murine target). Mice were euthanized 48 hours after the last dose, and organs and serum were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of HBV DNA, using primer probe sets RTS3370, RTS3371, or RTS3372 (forward sequence ATCCTATCAACACTTCCGGAAACT, designated SEQ ID NO: 314; reverse sequence CGACGCGGCGATTGAG, designated SEQ ID NO: 315; probe sequence AAGAACTCCCTCGCCTCGCAGACG, designated SEQ ID NO: 316). The DNA levels were normalized to picogreen. HBV RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 67. Serum DNA samples were analyzed after the study period. The data is presented in Table 68, expressed relative to the levels measured in the control group. As shown in Tables 67 and 68, the antisense oligonucleotides achieved reduction of HBV DNA and RNA over the PBS control. Results are presented as percent inhibition of HBV mRNA or DNA, relative to control. The Chemistry column defines the gap-wing structure of each gapmer.

TABLE 67 Percent inhibition of HBV RNA and DNA in the liver of transgenic mice % % % % % % inhibition inhibition inhibition inhibition inhibition inhibition ISIS DNA DNA DNA RNA RNA RNA No Chemistry (RTS3370) (RTS3371) (RTS3372) (RTS3370) (RTS3371) (RTS3372) 146786 5-10-5 MOE 97 97 95 86 85 89 510100 3-10-4 MOE 95 94 94 56 64 77 141923 5-10-5 MOE 2 0 13 0 7 31 459024 3-10-4 MOE 19 0 8 0 0 0

TABLE 68 Percent inhibition of HBV DNA in the serum of transgenic mice % inhibition % inhibition ISIS No (RTS3370) (RTS3371) 146786 98 98 510100 99 98 141923 0 0 459024 0 0 HBV antigen Analysts

HBV antigens in the supernatants were detected with the ELISA technique. HBs antigen (HBsAg) levels were detected by ELISA from Abazyme LLC, MA. As presented in Table 57, treatment with ISIS oligonucleotides 146786 or 510100 caused reduction in HBsAg levels. HBe antigen (HBeAg) levels were detected by ELISA from International Immuno-diagnostics, CA. As presented in Table 69, treatment with ISIS oligonucleotides 146786 or 510100 also caused reduction in HBeAg levels.

TABLE 69 HBV antigen levels (PEI U/mL) in transgenic mice HBsAg HBeAg PBS 40 80 146786 3 15 510100 15 22 141923 32 80 459024 44 51

Example 21 Antisense Inhibition of HBV Viral mRNA in HepG2 Cells by Deoxy, MOE and (S)-cEt Gapmers

Additional antisense oligonucleotides were designed targeting a HBV viral nucleic acid and were tested for their effects on HBV mRNA in vitro. ISIS 146786, ISIS 505358, ISIS 509932, and ISIS 510100, disclosed in U.S. Provisional Application No. 61/478,040 filed on Apr. 21, 2011; ISIS 552859 disclosed in U.S. Provisional Application No. 61/596,692 filed on Feb. 8, 2012; ISIS 577121, ISIS 577122, ISIS 577123, ISIS 577132, ISIS 577133, and ISIS 577134, disclosed in the study described above, were also included in the assay. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using Cytofectin with 9.375 nM, 18.75 nM, 37.50 nM, 75.00 nM, 150.00 nM, or 300.00 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3371 was used to measure mRNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of HBV, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in Tables below were designed as deoxy, MOE and (S)-cEt gapmers. The deoxy, MOE and (S)-cEt gapmers are 16, 17, or 18 nucleosides in length wherein the nucleosides have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; the number indicates the number of deoxynucleosides; otherwise, ‘d’ indicates a deoxynucleoside; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

“Viral Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted viral gene sequence. Each gapmer listed in Table 70 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1).

TABLE 70  Chimeric antisense oligonucleotides targeting SEQ ID NO: 1 Viral Viral Target Target SEQ ID Start Site Stop Site ISIS No Motif Sequence NO  411  427 585163 eeekk-8-eeee  GGCATAGCAGCAGGATG 17  414  430 585164 eeekk-7-kkeee TGAGGCATAGCAGCAGG 21  414  430 585165 eeek-9-keee TGAGGCATAGCAGCAGG 21 1577 1593 585170 eeekk-7-kkeee AAGCGAAGTGCACACGG 1304 1577 1593 585171 eeek-9-keee AAGCGAAGTGCACACGG 1304 1577 1593 585172 eeeekk-7-eeee   AAGCGAAGTGCACACGG 1304 1577 1593 585173 ekek-9-eeee AAGCGAAGTGCACACGG 1304 1577 1593 585174 ekekdk-7-eeee   AAGCGAAGTGCACACGG 1304 1583 1599 585166 eeekk-7-kkeee GAGGTGAAGCGAAGTGC 1310 1583 1599 585167 eeek-9-keee GAGGTGAAGCGAAGTGC 1310 1780 1797 577119 kdkdk-8-eeeee AATTTATGCCTACAGCCT 1379 1780 1796 585168 eeekk-7-kkeee ATTTATGCCTACAGCCT 51 1780 1796 585169 eeek-9-keee ATTTATGCCTACAGCCT 51

TABLE 71 Dose dependent inhibition of HBV mRNA levels by chimeric antisense oligonucleotides ISIS 9.375 18.75 37.5 75.0 150.0 300.0 No nM nM nM nM nM nM 146786 37 37 58 70 81 93 505358 30 26 28 57 74 85 510100 42 30 43 61 77 91 552859 21 30 39 61 79 91 577119 42 43 46 66 74 75 577121 10 15 42 64 82 89 577122 21 30 53 66 78 84 577123 27 29 45 56 78 84 577132 14 21 42 61 80 92 577133 12 14 32 47 62 77 577134 37 39 59 72 86 90 585174 31 28 48 61 80 90

TABLE 72 Dose dependent inhibition of HBV mRNA levels by chimeric antisense oligonucleotides ISIS 9.375 18.75 37.5 75.0 150.0 300.0 No nM nM nM nM nM nM 146786 25 34 57 71 85 92 509932 9 28 59 62 70 74 585163 17 32 52 68 77 81 585164 23 4 29 31 36 56 585165 6 31 42 58 66 82 585166 19 27 35 48 50 63 585167 22 25 50 69 76 88 585168 4 30 44 52 67 76 585169 32 32 42 62 76 80 585170 23 19 39 49 66 75 585171 28 27 42 59 81 88 585172 26 29 30 64 80 91 585173 29 30 41 71 86 88

Example 22 Analysis of the Potency of Uniform Deoxyoligonucleotides in Inhibition of HBV mRNA in HepG2 Cells

Additional antisense oligonucleotides were tested for their effects on HBV mRNA in vitro. ISIS 5808 and ISIS 9591, disclosed in U.S. Pat. No. 5,985,662 were also included in the assay. ISIS 146786 was included in the assay as the benchmark. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 18.75 nM, 37.50 nM, 75.00 nM, 150.00 nM, or 300.00 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HBV mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3371 was used to measure mRNA and DNA levels. HBV mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. S antigen and E antigen levels were also measured by ELISA. Results are presented as percent inhibition, relative to untreated control cells.

The antisense oligonucleotides tested, ISIS 582699, ISIS 582700, and ISIS 582701, were designed according to the sequences and chemistries disclosed in Korba and Gerin, Antiviral Research, 1995, Vol. 28, 225-242; the corresponding names for the oligonucleotides in the reference are S1, C1, and L2c, respectively. The antisense oligonucleotides in Tables below were designed as uniform deoxy oligonucleotides, 16 or 21 nucleosides in length wherein the nucleosides have deoxy modifications. “Viral Target start site” indicates the 5′-most nucleotide to which the oligonucleotide is targeted in the viral gene sequence. “Viral Target stop site” indicates the 3′-most nucleotide to which the oligonucleotide is targeted viral gene sequence. Each oligonucleotide listed in Table 73 is targeted to the viral genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. U95551.1). The results indicate that the deoxy oligonucleotides had negligible effect on HBV mRNA expression levels DNA levels and HBV antigen levels.

TABLE 73  Uniform deoxy oligonucleotides targeting SEQ ID NO: 1 Viral Viral Target Target SEQ Start Stop ISIS ID Site Site No Sequence NO  160  180 582699 GAATCCTGATGTGATGTTCTC 1378 1884 1899 582701 CCAAAGCCACCCAAGG 1380 1910 1930 582700 CAAATTCTTTATAAGGGTCGA 1381

TABLE 74 Dose dependent inhibition of HBV mRNA levels after treatment with oligonucleotides ISIS No 18.75 nM 37.5 nM 75.0 nM 150.0 nM 300.0 nM 5808 38 23 29 40 54 9591 35 20 32 26 40 146786 11 5 45 66 92 582699 32 28 27 39 52 582700 18 12 20 16 23 582701 4 0 0 3 13

TABLE 75 Dose dependent inhibition of HBV DNA levels in HepG2 cells after treatment with oligonucleotides ISIS No 18.75 nM 37.5 nM 75.0 nM 150.0 nM 5808 20 17 0 0 9591 0 0 0 0 146786 32 50 77 83 582699 0 44 0 17 582700 0 0 0 0 582701 0 0 0 0

TABLE 76 HBV S antigen levels after treatment with oligonucleotides (arbitrary units) ISIS No 18.75 nM 37.5 nM 75.0 nM 150.0 nM 5808 9,254 8,228 4,168 2,540 9591 10,924 8,683 9,334 12,142 146786 12,501 7,265 3,408 1,017 582699 9,340 9,325 7,589 4,712 582700 9,697 8,350 11,168 10,703 582701 15,283 18,209 14,632 15,299

TABLE 77 HBV E antigen levels after treatment with oligonucleotides (arbitrary units) ISIS No 18.75 nM 37.5 nM 75.0 nM 150.0 nM 5808 8,075 8,587 5,036 3,286 9591 9,242 8,093 8,257 6,944 146786 8,532 4,034 2,301 449 582699 7,815 7,191 7,026 5,278 582700 8,690 9,304 7,941 6,315 582701 8,847 8,257 8,211 6,276 

What is claimed is:
 1. A compound comprising a single-stranded modified oligonucleotide consisting of the sequence recited in any one of SEQ ID NOs: 17, 50, 722, 1327, 1340, and 1379, wherein the modified oligonucleotide is 100% complementary over its entire length with a nucleic acid encoding hepatitis B virus (HBV) and comprises: a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; and a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
 2. The compound of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
 3. The compound of claim 2, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.
 4. The compound of claim 1, wherein the modified sugar is a bicyclic sugar.
 5. The compound of claim 4, wherein the bicyclic sugar comprises a 4′-CH(CH3)-O-2′ group.
 6. The compound of claim 1, wherein the at least one modified sugar comprises a 2′-O-methoxyethyl group.
 7. The compound of claim 1, wherein at least one nucleoside comprises a modified nucleobase.
 8. The compound of claim 7, wherein the modified nucleobase is a 5-methylcytosine.
 9. The compound of claim 1, wherein the oligonucleotide consists of 17 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 17, and wherein the oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5′ wing segment consisting of 3 linked nucleosides; and a 3′ wing segment consisting of 4 linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage, and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.
 10. The compound of claim 1, wherein the oligonucleotide consists of 20 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 50, and wherein the oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5′ wing segment consisting of 5 linked nucleosides; and a 3′ wing segment consisting of 5 linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage, and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.
 11. The compound of claim 1, wherein the oligonucleotide consists of 20 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 722, and wherein the oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5′ wing segment consisting of 6 linked nucleosides; and a 3′ wing segment consisting of 4 linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage, and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.
 12. The compound of claim 1, wherein the oligonucleotide consists of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 1327, and wherein the oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5′ wing segment consisting of 2 linked nucleosides; and a 3′ wing segment consisting of 4 linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 2 linked nucleosides of the 5′ wing segment comprise a 2′-O-methoxyethyl sugar and a constrained ethyl sugar in the 5′ to 3′ direction; wherein the 4 linked nucleosides of the 3′ wing segment comprise a constrained ethyl sugar, a 2′-O-methoxyethyl sugar, a constrained ethyl sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage; and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.
 13. The compound of claim 1, wherein the oligonucleotide consists of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 1340, and wherein the oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5′ wing segment consisting of 3 linked nucleosides; and a 3′ wing segment consisting of 3 linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 3 linked nucleosides of the 5′ wing segment comprise a 2′-O-methoxyethyl sugar, a constrained ethyl sugar, and a constrained ethyl sugar in the 5′ to 3′ direction; wherein the 3 linked nucleosides of the 3′ wing segment comprise a constrained ethyl sugar, a constrained ethyl sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage; and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.
 14. The compound of claim 1, wherein the oligonucleotide consists of 18 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 1379, and wherein the oligonucleotide comprises: a gap segment consisting of 8 linked deoxynucleosides; a 5′ wing segment consisting of 5 linked nucleosides; and a 3′ wing segment consisting of 5 linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5 linked nucleosides of the 5′ wing segment comprise a constrained ethyl sugar, a deoxy sugar, a constrained ethyl sugar, a deoxy sugar, and a constrained ethyl sugar in the 5′ to 3′ direction; wherein each of the 5 linked nucleosides of the 3′ wing segment comprises a 2′-O-methoxyethyl sugar; wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage; and wherein each cytosine of the modified oligonucleotide is a 5-methylcytosine.
 15. A method of preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in an animal comprising administering to the animal the compound of claim 1, thereby preventing, treating, ameliorating, or slowing progression of a HBV-related disease, disorder or condition in the animal.
 16. The method of claim 15, wherein the disease, disorder or condition is jaundice, liver inflammation, liver fibrosis, inflammation, liver cirrhosis, liver failure, liver cancer, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, HBV viremia, or liver disease-related transplantation.
 17. The method of claim 16, wherein administering the compound of claim 1 reduces HBV antigen levels in the animal.
 18. The method of claim 17, wherein HBsAg levels are reduced.
 19. The method of claim 17, wherein HBeAg levels are reduced.
 20. The method of claim 15, wherein administering the compound of claim 1 induces HBsAG seroconversion in the animal. 